Virus compositions with enhanced specificity in the brain

ABSTRACT

Provided are compositions and kits comprising recombinant adeno-associated viruses (rAAVs) with tropisms showing increased specificity and efficiency of viral transduction in targeted cell-types, for e.g., the brain and the liver. Therapeutic and bio-medical research applications of the rAAVs are also described, including without limitation methods of discovering rAAVs using a multiplexed Cre recombination-based AAV targeted evolution (M-CREATE) method, and methods of treating various diseases and conditions by rAAV-mediated transgene therapy.

RELATED APPLICATIONS

This Application claims the benefit of and priority to U.S. ProvisionalPatent Application Ser. No. 62/832,836, filed Apr. 11, 2019, the contentof which is incorporated herein in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under NS087949,MH117069, and OD025535 awarded by National Institutes of Health. Thegovernment has certain rights in the invention.

SUMMARY

Recombinant adeno-associated viruses (rAAVs) are widely used as vectorsfor gene delivery in basic scientific research and therapeuticapplications because of their ability to transduce both dividing andnon-dividing cells, their long-term persistence as episomal DNA ininfected cells, and their low immunogenicity. These characteristics makethem appealing for applications in both basic science and in clinics,such as gene therapy. However, there is a need to significantly improvethe performance of existing serotypes to specifically target distinctbrain cell-types, upon systemic delivery to a subject. This need isespecially acute when the AAV must across the blood brain barrier (BBB)to reach the central nervous system (CNS).

Systemic delivery of existing AAV serotypes show limited transduction ofcertain cell types and organs, and non-specific, overlapping tropisms inothers. This leads to several complications in gene therapyapplications, including but not limited to off-target effects due totransduction of unimpacted organs and cell types (in particular, theliver), and the necessity for a larger viral dosage to achievesufficient therapeutic levels in the tissue or organ of interest.

Disclosed herein are rAAVs with engineered specificity into the capsidstructure through iterative rounds of positive and negative selection,yielding variants with tropisms having an increased specificity andtransduction efficiency when measured in the CNS, and in some cases, adecreased specificity and transduction efficiency in an off-targetenvironment, like the liver. The rAAVs described herein achievewidespread transduction to target environment (e.g., target cell typesor tissues) in a subject upon systemic delivery (e.g., intravenousinjection).

Also provided are methods of engineering the rAAVs of the presentdisclose using a multiplexed Cre recombination-based AAV targetedevolution (M-CREATE) method. The M-CREATE method generates enhancedtransduction efficiency and/or specificity by (1) introducing variationsin the capsid protein sequence, (2) in vivo unbiased selection andrecovery of only those variants that travel to defined cell populations,(3) cross the cell membrane, (4) travel to the nucleus, and (5)unpackage and express their genetic dosage amount. Variant capsidsexhibiting the most desirable tropism (e.g., enhanced efficiency andspecificity for a particular in vivo environment) are recovered andidentified by deep sequencing. Strategies for unbiased selection andanalysis include determining variants' enrichment score (by normalizingthe target tissue library to starting virus library) and unbiasedpropagation between rounds of selections through a synthetic libraryconstruction (where each variant is represented equally). Also disclosedare detailed characterizations of the resultant libraries fromsequencing data which provide useful insights on the selection ofvariants towards a target.

Disclosed herein are AAV capsid libraries generated using M-CREATE. Thefirst library, a 7-mer peptide insertion in AAV9 (7-mer-i library) wasbuilt for parallel in vivo selections across different brain celltypes—endothelial cells, neurons, and astrocytes —and yielded a largepool of AAV9 variants with enhanced ability to target as well as crossthe blood-brain barrier (BBB) and broadly transduce the central nervoussystem (CNS). The second library, a 3-mer peptide substitution inAAV-PHP.B β-mer-s library), was reinvestigated by incorporating deepsequencing to recover capsids. A pool of AAV-PHP.B variants werediscovered including a variant that transduce CNS neurons with greaterspecificity. The rAAVs of the present disclosure can efficiently targetthe endothelial cells of the blood-brain barrier, variants that canbroadly transduce different cell types in the central nervous system,and a variant exhibiting greater specificity towards transducing neuron.

Aspects disclosed herein provide AAV capsids comprising: (a) an AAVcapsid protein comprising: (i) a first amino acid sequence that is atleast 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1;and (ii) a second amino acid sequence at least 57.1% identical to anamino acid sequence provided in Tables 2-3 or FIG. 33 inserted at anamino acid position 588_589 within SEQ ID NO: 1, wherein the AAV capsidprotein is characterized by at least one of an increased specificity andan increased transduction efficiency when measured in a central nervoussystem (CNS) in a subject when delivered to the subject systemically,relative to a native AAV capsid protein provided in SEQ ID NO: 1. Insome embodiments, the second amino acid sequence is at least 71.4%identical to the amino acid sequence provided in Tables 2-3 or FIG. 33.In some embodiments, the second amino acid sequence is at least 86.7%identical to the amino acid sequence provided in Tables 2-3 or FIG. 33.In some embodiments, the second amino acid sequence is selected from thegroup consisting of TALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, SIERPFK,RYQGDSV, and TTLKPFS. In some embodiments, the AAV capsid protein ispresent in VP1, VP2, and VP3 of the AAV capsid. In some embodiments, theAAV capsid is chimeric. In some embodiments, 60 copies of the AAV capsidprotein are assembled into the AAV capsid. In some embodiments, the CNScomprises a cell-type selected from the group consisting of a neuron, anoligodendrocyte, an astrocyte, and a brain vascular cell. In someembodiments, the CNS comprises a tissue that is selected from the groupconsisting of a brain, a thalamus, a cortex, a striatum, a ventralmidbrain, and a spinal cord. In some embodiments, the AAV capsid proteinfurther comprises an amino acid substitution A587D. In some embodiments,the AAV capsid protein further comprises an amino acid substitutionQ588G. In some embodiments, the AAV capsid protein further comprises anamino acid substitution comprising A589N. In some embodiments, the AAVcapsid protein further comprises an amino acid substitution comprisingQ590P. In some embodiments, the second amino acid sequence at the aminoacid position 588_589 within SEQ ID NO: 1 is not TLAVPFK, KFPVALT,SVSKPFL, FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, or YTLSQGW. In someembodiments, the AAV capsid is isolated and purified. In someembodiments, the AAV capsid is formulated as a pharmaceuticalformulation for intravenous administration to treat a disease or acondition of the liver, the pharmaceutical formulation furthercomprising a pharmaceutically acceptable carrier. In some embodiments,the pharmaceutical formulation further comprises a therapeutic agent.

Aspects disclosed herein provide AAV capsids comprising an AAV capsidprotein comprising a seven amino acid insertion (X1 X2 X3 X4 X5 X6 X7)between amino acid 588 and amino acid 589 in an amino acid sequence ofthe AAV capsid protein provided in SEQ ID NO: 1, wherein X1 is an aminoacid selected from the group consisting of E, D, G, R, S and T. In someembodiments, X2 is an amino acid selected from the group consisting ofA, G, I, L, M, N, Q, T, and Y. In some embodiments, X3 is an amino acidselected from the group consisting of E, K, L, T, and Q. In someembodiments, X4 is an amino acid selected from the group consisting ofG, I, K, L, R, T, and V. In some embodiments, X5 is an amino acidselected from the group consisting of A, D, G, P, L, Q, and V. In someembodiments, X6 is an amino acid selected from the group consisting ofF, K, N, P, Q, S, and V. In some embodiments, X7 is an amino acidselected from the group consisting of I, K, L, P, and V. In someembodiments, the seven amino acid insertion is selected from the groupconsisting of TALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, SIERPFK, RYQGDSV, andTTLKPFS. In some embodiments, the AAV capsid protein is present in VP1,VP2, and VP3 of the AAV capsid. In some embodiments, the AAV capsid ischimeric. In some embodiments, 60 copies of the AAV capsid protein areassembled into the AAV capsid. In some embodiments, the AAV capsidprotein is characterized by at least one of an increased specificity andan increased transduction efficiency when measured in a central nervoussystem (CNS) in a subject when delivered to the subject systemically,relative to a native AAV capsid protein provided in SEQ ID NO: 1. Insome embodiments, the CNS comprises a cell-type selected from the groupconsisting of a neuron, a glial cell, a oligodendrocyte, an ependymalcell, an astrocyte, a Schwann cell, a satellite cell, and an entericglial cell. In some embodiments, the CNS comprises a tissue that isselected from the group consisting of a brain, a thalamus, a cortex, astriatum, a ventral midbrain, and a spinal cord. In some embodiments,the AAV capsid protein further comprises an amino acid substitutioncomprising A587D. In some embodiments, the AAV capsid protein furthercomprises an amino acid substitution comprising Q588G. In someembodiments, the AAV capsid protein further comprises an amino acidsubstitution comprising A589N. In some embodiments, the AAV capsidprotein further comprises an amino acid substitution comprising Q590P.In some embodiments, the seven amino acid insertion is not TLAVPFK,KFPVALT, SVSKPFL, FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, or YTLSQGW. Insome embodiments, the AAV capsid is isolated and purified. In someembodiments, the AAV capsid is formulated as a pharmaceuticalformulation for intravenous administration to treat a disease or acondition of the liver, the pharmaceutical formulation furthercomprising a pharmaceutically acceptable carrier. In some embodiments,the pharmaceutical formulation further comprises a therapeutic agent.

Aspects provided herein provide AAV capsids comprising: (a) an AAVcapsid protein comprising: (i) a first amino acid sequence that is atleast 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1;and (ii) a second amino acid sequence at least 57.1% identical to anamino acid sequence provided in Table 4 or FIG. 35 at an amino acidposition 588_589 within SEQ ID NO: 1, wherein the AAV capsid protein ischaracterized by at least one of an increased specificity and anincreased transduction efficiency when measured in a liver in a subjectwhen delivered to the subject systemically, relative to a native AAVcapsid protein provided in SEQ ID NO: 1. In some embodiments, the secondamino acid sequence is at least 71.4% identical to the amino acidsequence provided in Table 4 or FIG. 35. In some embodiments, the secondamino acid sequence is at least 86.7% identical to the amino acidsequence provided in Table 4 or FIG. 35. In some embodiments, the secondamino acid sequence is selected from the group consisting of KAYSVQV,PSGSARS, and RTANALG. In some embodiments, the AAV capsid protein ispresent in VP1, VP2, and VP3 of the AAV capsid. In some embodiments, theAAV capsid is chimeric. In some embodiments, 60 copies of the AAV capsidprotein are assembled into the AAV capsid. In some embodiments, the AAVcapsid is isolated and purified. In some embodiments, the AAV capsid isformulated as a pharmaceutical formulation for intravenousadministration to treat a disease or a condition of the liver, thepharmaceutical formulation further comprising a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical formulationfurther comprises a therapeutic agent.

Aspects disclosed herein provide AAV capsid proteins comprising: (i) afirst amino acid sequence that is at least 98% identical to amino acid217 to amino acid 736 of SEQ ID NO: 1; and (ii) a second amino acidsequence at least 57.1% identical to an amino acid sequence provided inTables 2-3 or FIG. 33 inserted at an amino acid position 588_589 withinSEQ ID NO: 1, wherein the AAV capsid protein is characterized by atleast one of an increased specificity and an increased transductionefficiency when measured in a central nervous system (CNS) in a subjectwhen delivered to the subject systemically, relative to a native AAVcapsid protein provided in SEQ ID NO: 1. In some embodiments, the secondamino acid sequence is at least 71.4% identical to the amino acidsequence provided in Tables 2-3 or FIG. 33. In some embodiments, thesecond amino acid sequence is at least 86.7% identical to the amino acidsequence provided in Tables 2-3 or FIG. 33. In some embodiments, thesecond amino acid sequence is selected from the group consisting ofTALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, SIERPFK, RYQGDSV, and TTLKPFS. Insome embodiments, the AAV capsid protein is present in VP1, VP2, and VP3of an AAV capsid. In some embodiments, the AAV capsid is chimeric. Insome embodiments, 60 copies of the AAV capsid protein are assembled intothe AAV capsid. In some embodiments, the CNS comprises a cell-typeselected from the group consisting of a neuron, an oligodendrocyte, anastrocyte, and a brain vascular cell. In some embodiments, the CNScomprises a tissue that is selected from the group consisting of abrain, a thalamus, a cortex, a striatum, a ventral midbrain, and aspinal cord. In some embodiments, the AAV capsid protein furthercomprises an amino acid substitution A587D. In some embodiments, the AAVcapsid protein further comprises an amino acid substitution Q588G. Insome embodiments, the AAV capsid protein further comprises an amino acidsubstitution comprising A589N. In some embodiments, the AAV capsidprotein further comprises an amino acid substitution comprising Q590P.In some embodiments, the second amino acid sequence at the amino acidposition 588_589 within SEQ ID NO: 1 is not TLAVPFK, KFPVALT, SVSKPFL,FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, or YTLSQGW. In some embodiments, theAAV capsid protein is isolated and purified. In some embodiments, theAAV capsid protein is formulated as a pharmaceutical formulation forintravenous administration to treat a disease or a condition of theliver, the pharmaceutical formulation further comprising apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical formulation further comprises a therapeutic agent.

Aspects disclosed herein provide AAV capsids proteins comprising a sevenamino acid insertion (X1 X2 X3 X4 X5 X6 X7) between amino acid 588 andamino acid 589 in an amino acid sequence of the AAV capsid proteinprovided in SEQ ID NO: 1, wherein X1 is an amino acid selected from thegroup consisting of E, D, G, R, S and T. In some embodiments, X2 is anamino acid selected from the group consisting of A, G, I, L, M, N, Q, T,and Y. In some embodiments, X3 is an amino acid selected from the groupconsisting of E, K, L, T, and Q. In some embodiments, X4 is an aminoacid selected from the group consisting of G, I, K, L, R, T, and V. Insome embodiments, X5 is an amino acid selected from the group consistingof A, D, G, P, L, Q, and V. In some embodiments, X6 is an amino acidselected from the group consisting of F, K, N, P, Q, S, and V. In someembodiments, X7 is an amino acid selected from the group consisting ofI, K, L, P, and V. In some embodiments, the seven amino acid insertionis selected from the group consisting of TALKPFL, TTLKPFL, TLQIPFK,TMQKPFI, SIERPFK, RYQGDSV, and TTLKPFS. In some embodiments, the AAVcapsid protein is present in VP1, VP2, and VP3 of a AAV capsid. In someembodiments, the AAV capsid is chimeric. In some embodiments, 60 copiesof the AAV capsid protein are assembled into the AAV capsid. In someembodiments, the AAV capsid protein is characterized by at least one ofan increased specificity and an increased transduction efficiency whenmeasured in a central nervous system (CNS) in a subject when deliveredto the subject systemically, relative to a native AAV capsid proteinprovided in SEQ ID NO: 1. In some embodiments, the CNS comprises acell-type selected from the group consisting of a neuron, a glial cell,a oligodendrocyte, an ependymal cell, an astrocyte, a Schwann cell, asatellite cell, and an enteric glial cell. In some embodiments, the CNScomprises a tissue that is selected from the group consisting of abrain, a thalamus, a cortex, a striatum, a ventral midbrain, and aspinal cord. In some embodiments, the AAV capsid protein furthercomprises an amino acid substitution comprising A587D. In someembodiments, the AAV capsid protein further comprises an amino acidsubstitution comprising Q588G. In some embodiments, the AAV capsidprotein further comprises an amino acid substitution comprising A589N.In some embodiments, the AAV capsid protein further comprises an aminoacid substitution comprising Q590P. In some embodiments, the seven aminoacid insertion is not TLAVPFK, KFPVALT, SVSKPFL, FTLTTPK, MNATKNV,NGGTSSS, TRTNPEA, or YTLSQGW. In some embodiments, the AAV capsidprotein is isolated and purified. In some embodiments, the AAV capsidprotein is formulated as a pharmaceutical formulation for intravenousadministration to treat a disease or a condition of the liver, thepharmaceutical formulation further comprising a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical formulationfurther comprises a therapeutic agent.

Aspects provided herein provide AAV capsids proteins comprising: (i) afirst amino acid sequence that is at least 98% identical to amino acid217 to amino acid 736 of SEQ ID NO: 1; and (ii) a second amino acidsequence at least 57.1% identical to an amino acid sequence provided inTable 4 or FIG. 35 at an amino acid position 588_589 within SEQ ID NO:1, wherein the AAV capsid protein is characterized by at least one of anincreased specificity and an increased transduction efficiency whenmeasured in a liver in a subject when delivered to the subjectsystemically, relative to a native AAV capsid protein provided in SEQ IDNO: 1. In some embodiments, the second amino acid sequence is at least71.4% identical to the amino acid sequence provided in Table 4 or FIG.35. In some embodiments, the second amino acid sequence is at least86.7% identical to the amino acid sequence provided in Table 4 or FIG.35. In some embodiments, the second amino acid sequence is selected fromthe group consisting of KAYSVQV, PSGSARS, and RTANALG. In someembodiments, the AAV capsid protein is present in VP1, VP2, and VP3 ofan AAV capsid. In some embodiments, the AAV capsid is chimeric. In someembodiments, 60 copies of the AAV capsid protein are assembled into theAAV capsid. In some embodiments, the AAV capsid protein is isolated andpurified. In some embodiments, the AAV capsid protein is formulated as apharmaceutical formulation for intravenous administration to treat adisease or a condition of the liver, the pharmaceutical formulationfurther comprising a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical formulation further comprises atherapeutic agent.

Aspects disclosed herein comprise plasmid vectors comprising a nucleicacid sequence encoding the AAV capsids and AAV capsid proteins describedherein. In some instances, the plasmid vector is bacterial. In someinstances, the plasmid vector is derived from Escherichia coli. In someinstances, the nucleic acid sequence comprises, in a 5′ to 3′ direction:(1) a 5′ inverted terminal repeat (ITR) sequence, (2) a Replication(Rep) gene, (3) a Capsid (Cap) gene, and (4) a 3′ ITR, wherein the Capgene encodes the AAV capsid protein described herein. In some instances,the plasmid vector encodes a pseudotyped AAV capsid protein. In someinstances, the Cap gene is derived from the deoxyribose nucleic acid(DNA) provided in any one of SEQ ID NOs: 6-10. In some instances, thenucleic acid sequence comprising the Cap gene is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of theDNA sequences provided in U.S. App. Ser. No. 16/582,635, incorporatedherein by reference. In some instances, the 5′ ITR and the 3′ ITR arederived from an AAV2 serotype. In some instances, the 5′ ITR and the 3′ITR are derived from an AAVS serotype. In some instances, the 5′ ITR andthe 3′ ITR are derived from an AAV9 serotype.

Aspects disclosed herein provide methods of treating a disease orcondition in a subject comprising administering a therapeuticallyeffective amount of a pharmaceutical formulation comprising the AAVcapsid protein or the AAV capsid of the present disclosure. In someembodiments, the disease or the condition is a disease or a condition ofa central nervous system (CNS) or a liver of the subject. In someembodiments, the disease or condition of the liver is selected from thegroup consisting of Alagille Syndrome, Alcohol-Related Liver Disease,Alpha-1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign LiverTumors, Biliary Atresia, Cirrhosis, Crigler-Najjar Syndrome,Galactosemia, Gilbert Syndrome, Hemochromatosis, Hepatic Encephalopathy,Hepatitis A, Hepatitis B, Hepatitis C, Hepatorenal Syndrome,Intrahepatic Cholestasis of Pregnancy (ICP), Lysosomal Acid LipaseDeficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice,Non-Alcoholic Fatty Liver Disease, Primary Biliary Cholangitis (PBC),Primary Sclerosing Cholangitis (PSC), Reye Syndrome, Type I GlycogenStorage Disease, and Wilson Disease. In some embodiments, the disease orcondition of the CNS is selected from group consisting of Absence of theSeptum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency,Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis,Attention Deficit-Hyperactivity Disorder (ADHD), Adie's Pupil, Adie'sSyndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum,Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS-Neurological Complications, Alexander Disease, Alpers' Disease,Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic LateralSclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis,Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts,Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation,Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellaror Spinocerebellar Degeneration, Atrial Fibrillation and Stroke,Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorder,Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease,Becker's Myotonia, Behcet's Disease, Bell's Palsy, Benign EssentialBlepharospasm, Benign Focal Amyotrophy, Benign IntracranialHypertension, Bernhardt-Roth Syndrome, Binswanger's Disease,Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus BirthInjuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brainand Spinal Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome,Bulbospinal Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathywith Subcortical Infarcts and Leukoencephalopathy (CADASIL), CanavanDisease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, CavernousAngioma, Cavernous Malformation, Central Cervical Cord Syndrome, CentralCord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis,Cephalic Disorders, Ceramidase Deficiency, Cerebellar Degeneration,Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis,Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavemous Malformation,Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy,Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-ToothDisease, Charcot-Marie-Tooth syndrome, classical rhizomelicchondrodysplasia punctata (RCDP), Chiari Malformation, Cholesterol EsterStorage Disease, Chorea, Choreoacanthocytosis, Chronic InflammatoryDemyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance,Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome,Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital FacialDiplegia, Congenital Myasthenia, Congenital Myopathy, CongenitalVascular Cavernous Malformations, Corticobasal Degeneration, CranialArteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt- JakobDisease, Cumulative Trauma Disorders, Cushing's Syndrome, CytomegalicInclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-DancingFeet Syndrome, Dandy-Walker Syndrome, Dawson Disease, Deafness, DeMorsier's Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia -Multi-Infarct, Dementia—Semantic, Dementia—Subcortical, Dementia With LewyBodies, Dentate Cerebellar Ataxia, Dentatorubral Atrophy,Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, DiabeticNeuropathy, Diffuse Sclerosis, Dravet Syndrome, Duchenne musculardystrophy, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia,Dyssynergia Cerebellaris Myoclonica, Dyssynergia CerebellarisProgressiva, Dystonias, Early Infantile Epileptic Encephalopathy, EmptySella Syndrome, Encephalitis, Encephalitis Lethargica, Encephaloceles,Encephalopathy, Encephalopathy (familial infantile), EncephalotrigeminalAngiomatosis, Epilepsy, Epileptic Hemiplegia, Erb's Palsy, Erb-Duchenneand Dejerine-Klumpke Palsies, Essential Tremor, ExtrapontineMyelinolysis, Fabry Disease, Fahr's Syndrome, Fainting, FamilialDysautonomia, Familial Hemangioma, Familial Idiopathic Basal GangliaCalcification, Familial Periodic Paralyses, Familial Spastic Paralysis,Farber's Disease, Febrile Seizures, Fibromuscular Dysplasia, FisherSyndrome, Floppy Infant Syndrome, Foot Drop, Friedreich's Ataxia,Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidoses,Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, GiantAxonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease,glioblastoma, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia,Glycogen Storage Disease, Guillain-Barre Syndrome, Hallervorden-SpatzDisease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm,Hemiplegia Alterans, Hereditary Neuropathies, Hereditary SpasticParaplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster,Herpes Zoster Oticus, Hirayama Syndrome, Holmes-Adie syndrome,Holoprosencephaly, HTLV-1 Associated Myelopathy, Hughes Syndrome,Huntington's Disease, Hydranencephaly, Hydrocephalus,Hydrocephalus—Normal Pressure, Hydromyelia, Hypercortisolism,Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-MediatedEncephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti,Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile PhytanicAcid Storage Disease, Infantile Refsum Disease, Infantile Spasms,Inflammatory Myopathies, Iniencephaly, Intestinal Lipodystrophy,Intracranial Cysts, Intracranial Hypertension, Isaacs' Syndrome, JoubertSyndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome,Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome(KTS), Kliiver-Bucy Syndrome, Korsakoff s Amnesic Syndrome, KrabbeDisease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton MyasthenicSyndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous NerveEntrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh'sDisease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy,Levine-Critchley Syndrome, Lewy Body Dementia, Lipid Storage Diseases,Lipoid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig'sDisease, Lupus -Neurological Sequelae, Lyme Disease—NeurologicalComplications, Machado—Joseph Disease, Macrencephaly, Megalencephaly,Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis,Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy,Microcephaly, Migraine, Miller Fisher Syndrome, Mini Stroke,Mitochondrial Myopathy, Moebius Syndrome, Monomelic Amyotrophy, MotorNeuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses,Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis,Multiple System Atrophy, Multiple System Atrophy with OrthostaticHypotension, Muscular Dystrophy, Myasthenia -Congenital, MyastheniaGravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy ofInfants, Myoclonus, Myopathy, Myopathy- Congenital, Myopathy-Thyrotoxic, Myotonia, Myotonia Congenita, Narcolepsy,Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation,Neurofibromatosis, Neuroleptic Malignant Syndrome, NeurologicalComplications of AIDS, Neurological Complications of Lyme Disease,Neurological Consequences of Cytomegalovirus Infection, NeurologicalManifestations of Pompe Disease, Neurological Sequelae Of Lupus,Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis,Neuronal Migration Disorders, Neuropathy- Hereditary, Neurosarcoidosis,Neurosyphilis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease,O′Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome,Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, OrthostaticHypotension, Overuse Syndrome, Pain -Chronic, PantothenateKinase-Associated Neurodegeneration, Paraneoplastic Syndromes,Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis, ParoxysmalHemicrania, Parry -Romberg, Pelizaeus-Merzbacher Disease, Pena ShokeirII Syndrome, Perineural Cysts, Periodic Paralyses, PeripheralNeuropathy, Periventricular Leukomalacia, Persistent Vegetative State,Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick'sDisease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors,Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome,Postherpetic Neuralgia, Postinfectious Encephalomyelitis, PosturalHypotension, Postural Orthostatic Tachycardia Syndrome, PosturalTachycardia Syndrome, Primary Dentatum Atrophy, Primary LateralSclerosis, Primary Progressive Aphasia, Prion Diseases, ProgressiveHemifacial Atrophy, Progressive Locomotor Ataxia, Progressive MultifocalLeukoencephalopathy, Progressive Sclerosing Poliodystrophy, ProgressiveSupranuclear Palsy, Prosopagnosia, Pseudo-Torch syndrome,Pseudotoxoplasmosis syndrome, Pseudotumor Cerebri, Psychogenic Movement,Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen'sEncephalitis, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease,Refsum Disease—Infantile, Repetitive Motion Disorders, Repetitive StressInjuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, RettSyndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome,Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease,Sandhoff Disease, Schilder's Disease, Schizencephaly, SeitelbergerDisease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia,Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome,Shingles, Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, SleepingSickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal CordInfarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal MuscularAtrophy, Spinocerebellar Atrophy, Spinocerebellar Degeneration,Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome,Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, SubacuteSclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy,Short-lasting, Unilateral, Neuralgiform (SUNCT) Headache, SwallowingDisorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis,Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, TabesDorsalis,Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, TemporalArteritis, Tethered Spinal Cord Syndrome, Thomsen's Myotonia, ThoracicOutlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis,Tourette Syndrome, Transient Ischemic Attack, Transmissible SpongiformEncephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor,Trigeminal Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome,Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis Syndromes of theCentral and Peripheral Nervous Systems, Von Economo's Disease, VonHippel-Lindau Disease (VHL), Von Recklinghausen's Disease, Wallenberg'sSyndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, WestSyndrome, Whiplash, Whipple's Disease, Williams Syndrome, WilsonDisease, Wolman's Disease, and X-Linked Spinal and Bulbar MuscularAtrophy. In some embodiments, the pharmaceutical formulation comprises atherapeutic nucleic acid encoding a therapeutic gene expression product.In some instances, the therapeutic gene expression product is effectiveto modulate an activity or an expression of a target gene or geneexpression product selected from the group consisting of SarcoglycanAlpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic aciddecarboxylase 67 (GAD67), CLN2, Nerve Growth Factor (NGF), Survival OfMotor Neuron 1, Telomeric (SMN1), Factor X (FIX), RetinoidIsomerohydrolase (RPE65), sarco/endoplasmic reticulum Ca2+-ATPase(SERCA2a), β-Glucocerebrosidase (GCase), Frataxin (FXN), Huntingtin(HTN), methyl-CpG binding protein 2 (MECP2), a peroxisomal biogenesisfactor (PEX), progranulin (GRN), an antitubulin agent, copper-zincsuperoxide dismutase (SOD1), Glucosylceramidase Beta (GBA), NPCIntracellular Cholesterol Transporter 1 (NPC1), and a NLRP3inflammasome. In some instances, the therapeutic gene expression productcomprises gene editing components. In some instances, the gene editingcomponents are selected from the group consisting of small interferingRNA (siRNA), short hairpin RNA (shRNA), a microRNA (miRNA), artificialsite-specific RNA endonuclease (ASRE), zinc finger endonuclease (ZFN),CRISPR/Cas, and transcription factor like effector nuclease (TALEN).

Aspects disclosed herein provide methods of manufacturing a recombinantAAV particle from the AAV capsid of the present disclosure, the methodcomprising: (a) introducing into a cell a nucleic acid comprising: (i) afirst nucleic acid sequence encoding a therapeutic gene expressionproduct; (ii) a second nucleic acid sequence encoding a recombinantviral genome comprising a capsid (Cap) gene modified to express the AAVcapsid of the present disclosure; and (iii) a third nucleic acidsequence encoding an AAV helper virus genome; and (b) assembling therecombinant AAV particle, the recombinant AAV particle comprising theAAV capsid encapsidating the first nucleic acid.

Aspects disclosed herein provide methods of manufacturing comprising:(a) introducing into a cell a nucleic acid comprising: (i) a firstnucleic acid sequence encoding a therapeutic gene expression productenclosed by a 5′ and a 3′ inverted terminal repeat (ITR) sequence; (ii)a second nucleic acid sequence encoding a viral genome comprising a 5′ITR sequence, a Replication (Rep) gene, Capsid (Cap) gene, and a 3′ ITR,wherein the Cap gene encodes the AAV capsid protein described herein;and (iii) a third nucleic acid sequence encoding a first helper virusprotein selected from the group consisting of E4orf6, E2a, and VA RNA,and optionally, a second helper virus protein comprising Ela or E1b55k;(b) expressing in the cell the AAV capsid protein described herein; (c)assembling an AAV particle comprising the AAV capsid proteins disclosedherein; and (d) packaging the first nucleic acid sequence in the AAVparticle. In some instances, the cell is mammalian. In some instances,the cell is immortalized. In some instances, the immortalized cell is anembryonic stem cell. In some instances, the embryonic stem cell is ahuman embryonic stem cell. In some instances, the human embryonic stemcell is a human embryonic kidney 293 (HEK-293) cell. In some instances,the Cap gene is derived from the deoxyribose nucleic acid (DNA) providedin any one of SEQ ID NOs: 6-10. In some instances, the nucleic acidsequence comprising the Cap gene is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the DNAsequences provided in U.S. App. Ser. No. 16/582,635, incorporated hereinby reference. In some instances, the 5′ ITR and the 3′ ITR are derivedfrom an AAV2 serotype. In some instances, the 5′ ITR and the 3′ ITR arederived from an AAVS serotype. In some instances, the 5′ ITR and the 3′ITR are derived from an AAV9 serotype. In some instances, the firstnucleic acid sequence and the second nucleic acid sequence are in trans.In some instances, the first nucleic acid sequence and the secondnucleic acid sequence are in cis. In some instances, the first nucleicacid sequence, the second nucleic acid sequence and the third nucleicacid sequence, are in trans.

Aspects disclosed herein provide methods of manufacturing a recombinantAAV particle, the method comprising: (a) providing a recombinant AAVgenome comprising: (i) an AAV capsid gene, and (ii) a recognitionsequence for a Cre recombinase, wherein the recognition sequencefacilitates a recombinase-dependent change that is detectable, andwherein the recombinase recognition sequence comprises twoCre-recognition sites; (iii) transfecting a population of cellsexpressing the Cre recombinase with the recombinant AAV genome, wherebythe Cre recombinase induces a recombination event to generate therecombinase-dependent change in the recombinant AAV genome, and whereinthe recombinase-dependent change comprises an inversion of the sequencethat is flanked by the Cre-recognition sites; (iv) detecting anincreased rate of the recombinase-dependent change a target cell in thepopulation of cells; (v) detecting a decreased rate of therecombinase-dependent change in an off-target cell in the population ofcells; and (vi) identifying a recombinant AAV genome generated by therecombinase-dependent change, wherein said identified rAAV genomecomprises the inversion, and wherein said identified recombinant AAVgenome encodes an AAV capsid particle characterized having an increasedspecificity for the target cell and a decreased specificity for theoff-target cell. In some embodiments, the off-target cell is ahepatocyte. In some embodiments, the target cell is a cell selected fromthe group consisting of a neuron, a glial cell, a oligodendrocyte, anependymal cell, an astrocyte, a Schwann cell, a satellite cell, and anenteric glial cell.

Aspects disclosed herein provide kits comprising: (a) a first vectorcomprising the recombinant vector of the present disclosure; (b) asecond vector encoding a helper virus protein; and (c) a third vectorcomprising a therapeutic nucleic acid encoding a therapeutic geneexpression product.

Aspects disclosed herein provide kits comprising: (a) a first vectorcomprising a first nucleic acid sequence encoding a viral genomecomprising in a 5′ to 3′ direction: (i) a 5′ inverted terminal repeat(ITR) sequence; (ii) a Replication (Rep) gene; (iii) a Capsid (Cap) geneencoding the AAV capsid proteins described herein, and (iv) a 3′ ITR;and (b) optionally, a second vector comprising a second nucleic acidsequence encoding a helper virus protein comprising at least one ofE4orf6, E2a, VA RNA, Ela and E1b55k. In some instances, the kit furthercomprises a cell. In some instances, the cell is mammalian. In someinstances, the cell is immortalized. In some instances, the immortalizedcell is an embryonic stem cell. In some instances, the embryonic stemcell is a human embryonic stem cell. In some instances, the humanembryonic stem cell is a human embryonic kidney 293 (HEK-293) cell. Insome instances, the kit further comprises an AAV vector comprising aheterologous nucleic acid encoding a therapeutic gene expressionproduct. In some instances, the AAV vector is an episome.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a multiplexed selection approach to identify capsids withspecific and broad tropisms. Steps 1-6 describe the workflow in Round-1(R1) selection, steps 7-9 describe Round-2 (R2) selection usingsynthetic pool method, steps la, 2a, and 6a-b show the incorporation ofdeep sequencing to recover capsids after R1 and R2 selection, and steps10-11 describe positive and/or negative selection criteria followed byvariant characterization.

FIG. 2 shows a structural model of the AAV9 capsid (PDB 3UX1) with theinsertion site for the 7-mer-i library highlighted in red in the60-meric (left), trimeric (middle), and monomeric (right) forms.

FIG. 3 shows Empirical Cumulative Distribution Frequency (ECDF) of R1DNA and virus libraries that were recovered by deep sequencing postGibson assembly and virus production, respectively.

FIG. 4 shows distributions of variants recovered from three R1 librariesfrom Tek-Cre, SNAP25-Cre, and GFAP-Cre brain tissue (n=2 per Cre line),with capsid libraries sorted by decreasing order of the enrichmentscore. The enrichment scores of the AAV-PHP.V2 variant are mapped aswell.

FIG. 5 is a schematic of R2 synthetic pool (left) and PCR pool (right)library design.

FIG. 6 shows an overlapping bar chart showing the percentage of libraryoverlap between the mentioned libraries and their theoreticalcomposition.

FIG. 7 shows histograms of DNA and virus libraries from the two methods,where the variants in a library are binned by their read counts (inlog10 scale) and the height of the histogram is proportional to theirfrequency.

FIG. 8 shows distributions of R2 brain libraries from all Cre transgeniclines (n=2 mice per Cre Line, mean is plotted) and both methods, wherethe libraries are sorted in decreasing order of enrichment score (log10scale). The total number of positively enriched variants from theselibraries are highlighted by dotted straight lines and AAV9's relativeenrichment is mapped on the synthetic pool plot.

FIG. 9 shows a comparison of the enrichment scores (log10 scale) of twoalternate codon replicates for 8462 variants from the Tek-Cre brainlibrary (n=2 mice, mean is plotted). The broken line separates thehigh-confidence signal (>0.3) from noise. For the high-confidence signal(below), a linear least-squares regression is determined between the 2codons and the regression line (best fit). The coefficient ofdetermination R2 is shown.

FIG. 10 shows heatmaps representing the magnitude (log2 fold change) ofa given AA's relative enrichment or depletion at each position givenstatistical significance is reached (boxed if P-value <0.0001,two-sided, two-proportion z-test, p-values corrected for multiplecomparisons using Bonferroni correction). R2 DNA normalized to oligopool(top, ˜9000 AA sequences), R2 virus normalized to R2 DNA (middle,n=˜9000 sequences), R2 Tek brain library with enrichment over 0.3(high-confidence signal) from synthetic pool method normalized to R2virus (bottom, 154 sequences) (n=2 for brain library, one per mouse. Allother libraries, n=1).

FIG. 11 shows heatmaps of Cre-independent relative enrichment acrossorgans (n=2 mice per Cre line, mean across 6 samples from 3 Cre lines isplotted) for variants enriched in the brain tissue of at least oneCre-dependent synthetic pool selection (lighter text, n=2 mice percell-type, mean is plotted) (left). Zoom-in of the most CNS-enrichedvariants (middle), and of the variants that are characterized in thecurrent study along with spike-in library controls (right) are shown.

FIG. 12 shows clustering analysis of variants from synthetic pool brainlibraries after enrichment in Tek-Cre (left), GFAP-Cre (middle), andcombined SNAP-Cre and Syn-Cre (right) selections. Size of nodesrepresents relative enrichment in brain. Thickness of edges (connectinglines) representing degree of relatedness. Distinct families (yellow)with the corresponding AA frequency logos (AA size represents prevalenceand color encodes AA properties).

FIG. 13 shows the 7-mer insertion peptide sequences of AAV-PHP variantsbetween AA positions 588-589 of AAV9 capsid.

FIG. 14 shows AAV9 (left) and AAV-PHP.V1 (right) mediated expressionusing ssAAV:CAG-mNeongreen genome (n=3, 3 weeks of expression inC57BL/6J adult mice with 3×10¹¹ vg IV dose/mouse). The images wereacquired under same microscope settings as that of the sagittal sectionsof brain (top) with higher magnification image from cortex (bottom).aGLUT1 antibody staining was used in the image to show vasculature.

FIG. 15 shows vascular transduction by ssAAV-PHP.V1:CAG-DIO-EYFP inTek-Cre adult mice (left) (n=2, 4 weeks of expression, 1×10¹² vg IVdose/mouse), and by ssAAV-PHP.V1:Ple261-iCre in Ai14 reporter mice(right) (n=2, 3 weeks of expression, 3×10¹¹ vg IV dose/mouse). Tissuesare stained with aGLUT1.

FIG. 16 shows percentage of vasculature stained with aGLUT1 thatoverlaps with mNeongreen (XFP) expression in cortex. One-way ANOVAnon-parametric Kruskal-Wallis test (P-value 0.0036), and follow-upmultiple comparisons using uncorrected Dunn's test (P-value of 0.0070for AAV9 vs PHP.V1) are reported. **P <0.01 is shown, P >0.05 is notshown; data is mean ±S.E.M, n=3 mice per AAV variant, cells quantifiedfrom 2-4 images per mouse per cell-type.

FIG. 17 shows percentage of cells stained with each cell-type specificmarker (aGLUT1, aS100 for astrocytes, aNeuN for neurons, aOlig2 foroligodendrocyte lineage cells) that overlaps with mNeongreen (XFP)expression in cortex. Kruskal-Wallis test (P-value of 0.0078), anduncorrected Dunn's test (P-value of 0.0235 for neuron vs vascular cells,and 0.0174 for neuron vs astrocyte, respectively) are reported. *P 0.05is shown, and P >0.05 is not shown; data is mean ±S.E.M, n=3 mice, cellsquantified from 2-4 images per mouse per cell-type.

FIG. 18 shows efficiency of vascular transduction (as described in FIG.16) in Tek-Cre mice (n=2, mean from 3 images per mouse per brainregion).

FIG. 19 shows efficiency of vascular transduction in Ai14 mice (n=2, amean from 4 images per mouse per brain region).

FIG. 20 shows transduction by AAV-PHP.B4-B6 and Cl variants, as well asB, eB, and AAV9 controls in sagittal brain and liver sections. Acrosseach set of images (column-wise) acquired, microscope settings werematched with AAV-PHP.eB. The white box on the sagittal brain imagesmarks the thalamus and not the precise region of the figures to theright. Vectors are packaged with ssAAV:CAG-2xNLS-EGFP genome (n=3 pergroup, lx10¹¹ vg IV dose/adult C57BL/6J mouse, 3 weeks of expression).Tissues are stained with cell-type specific markers: aNeuN for neurons,aS100 for astrocytes and aOlig2 for oligodendrocyte lineage cells. Livertissues are stained with a DNA stain, DAPI.

FIGS. 21-23 show the percentage of aNeuN+(FIG. 21), aS100+(FIG. 22), andaOlig2+(FIG. 23) cells with detectable nuclear-localized EGFP in theindicated brain regions (n=3 per group, lx10¹¹ vg dose). A two-way ANOVAwith correction for multiple comparisons using Tukey's test is reportedwith adjusted P-values (****P <0.0001, ***P <0.001, **P <0.01, *P <0.05,is shown, and P >0.05 is not shown on the plot; 95% CI, data is mean±S.E.M. The dataset comprises a mean of 2 images per region percell-type marker per mouse).

FIG. 24 shows the design of the 3-mer-s PHP.B library with combinationsof three AA diversification between AA 587-597 of AAV-PHP.B (orcorresponding AA 587-590 of AAV9). Shared AA identity with the parentAAV-PHP.B is shown along with unique motifs for AAV-PHP.N andAAV-PHP.eB.

FIG. 25 shows distributions of R2 brain and liver libraries (at AAlevel) by enrichment score (normalized to R2 virus library, withvariants sorted in decreasing order of enrichment score). The enrichmentof AAV-PHP.eB and AAV-PHP.N across all libraries are mapped on the plot.

FIG. 26 shows heatmap represents the magnitude (log2 fold change) of agiven AA's relative enrichment or depletion at each position across thediversified region, only if statistical significance is reached on foldchange (boxed if p-value <0.0001, two-sided, two-proportion z-test,p-values corrected for multiple comparisons using Bonferronicorrection). Plot includes variants that were highly enriched in brain(>0.5 mean enrichment score, where mean is drawn across Vglut2, Vgat andGFAP, n=1 library per mouse line (sample pooled from 2 mice per line))and underrepresented in liver (<0.0) (32 AA sequences).

FIG. 27 shows clustering analysis of enriched variants from Vgat brainlibrary with node size representing the degree of negative enrichment inliver and the thickness of edges (connecting lines) representing degreeof relatedness between nodes. Two distinct families are highlighted inyellow and their corresponding AA frequency logos are shown below (AAsize represents prevalence and color encodes AA properties).

FIG. 28 shows the percentage of neurons, astrocytes and oligodendrocytelineage cells with ssAAV-PHP.N:CAG-2xNLS-EGFP in the indicated brainregions (n=3, lx10¹¹ vg IV dose per adult C57BL/6J mouse, 3 weeks ofexpression, data is mean±S.E.M, 6-8 images for cortex, thalamus andstriatum, and 2 images for ventral midbrain, per mouse per cell-typemarker using 20x objective covering the entire regions). A two-way ANOVAwith correction for multiple comparisons using Tukey's test gaveadjusted P-values reported as ****P <0.0001, ns for P >0.05, 95% CI.

FIG. 29 shows transduction by ssAAV-PHP.N:CAG-NLS-EGFP (n=2, 2×10¹¹ vgIV dose per adult C57BL/6J mouse, 3 weeks of expression) with NeuNstaining (magenta) across three brain areas (cortex, SNc (substantianigra pars compacta) and thalamus).

FIG. 30 shows clustering analysis showing the brain-enriched sequencefamilies of all variants described herein, either identified in priorstudies (PHP.B-B3, PHP.eB) or in the current study (PHP.B4-B8, PHP.V1-2,PHP.C1-3). The thickness of edges (connecting lines) representing degreeof relatedness between nodes. The AA sequences inserted between 588-589(of AAV9 capsid) for all the variants discussed are shown below.

FIG. 31 shows transduction of AAV9, AAV-PHP.V1 and AAV-PHP.N acrossthree different mouse strains: C57BL/6J, BALB/cJ and FVB/NJ in sagittalbrain sections (right), along with a higher magnification image of thethalamus brain region (left).

FIG. 32 shows transduction by AAV-PHP.B, AAV-PHP.C1-C3 in C57BL/6J andBALB/cJ mice in sagittal brain sections (right), along with a highermagnification image of the thalamus brain region (left). In FIGS. 31 and32, the white box on the sagittal brain images represents the locationof thalamus and not the precise area that is zoomed-in on the figure tothe left. The microscope settings of acquired images were matched acrossall sagittal sections and across all thalamus regions. The insets inAAV-PHP.V1 are zoom-ins with enhanced brightness. The indicated capsidswere used to package ssAAV:CAG-mNeongreen (n=2-3 per group, 1×10¹¹ vg IVdose per 6-8 weeks old adult mouse, 3 weeks of expression. The datareported in FIGS. 31 and 32 are from one independent trial where allviruses were freshly prepared and titered in the same assay for dosageconsistency. AAV-PHP.C2 and AAV-PHP.C3 were further validated in anindependent trial for BALB/cJ, n=2 per group).

FIG. 33 provides 7-mer and 11-mer variants that were positively enrichedin the brain tissue in all cell lines.

FIG. 34 provides 7-mer and 11-mer variants enriched in one specificlibrary, but negatively enriched in all other brain and liver libraries.

FIG. 35 provides 11-mer variants that were positively enriched in allcre lines in liver tissue.

FIG. 36 is a diagram of the genetic switch used in M-CREATE. TheAcceptor Vector shows the position of the forward and reverse primersbetween the Lox sites that are used for selective recovery of capsidsfrom the Cre+cells. The Rep-AAPACap vector shows a deletion of 480 bp inCap gene in addition to the stop codons that are designed to preventsynthesis of VP1, VP2, and VP3 proteins. AAP protein translation isunaffected by these modifications.

FIG. 37 is a schematic of the protocol to selectively recover rAAVgenomes from the target population using the Cre-Lox flipping strategyand preparation of the sample for deep sequencing.

FIG. 38 illustrates the library coverage for R1 DNA and virus librariesobtained from specific sequencing depths.

FIG. 39 shows the percentage of variant overlap within the sampled DNAand virus, or across different Cre lines within tissues, or acrosstissues from R1 selection.

FIG. 40 shows the distributions of AAV capsid read counts for librariesrecovered by NGS from brain tissue across different Cre transgenic micepost R1 selection. The dotted line is illustrative only and roughlyseparates the signal from noise (see Methods for estimation of signalv.s. noise) where signal in this context represents the input for the R2selection.

FIG. 41 shows rAAV genome recovery from tissues using differenttreatments are shown with total rAAV genome recovery from 0.1 g ofliver.

FIG. 42 shows the percentage of rAAV genomes recovered per ng of totalextracted DNA.

FIG. 43 shows the CT value (cycle threshold from qPCR) of rAAV genomeextracted by trizol that were treated with Smal restriction enzyme oruntreated.

FIG. 44 shows CT value of mitochondrial DNA (internal control forsmaller genome recovery, fold change =10.79 (2ACT)) recovered from 1 ngof total DNA from liver tissue. In FIGS. 41-44, n=4 mice; 2 fromGFAP-Cre line and 2 from Tek-Cre line, each data point is drawn from themean of three technical replicates, error bar is mean±S.E.M.,Mann-Whitney test, two-tailed (exact P-value of 0.0286 (*P <0.05), inFIGS. 41, 42, and 44, and 0.1143 (n.s., P >0.05, CI 95%) in FIG. 43).The data reported FIGS. 41, 42, and 44 are from one independent trial,and FIG. 43, from three independent trials.

FIG. 45 shows the vector yields obtained per 10 ng of capsid DNA libraryacross R1 and R2 vector productions.

FIG. 46 shows distributions of the DNA and virus libraries produced bythe synthetic pool and PCR pool methods by the standard score of NGSread counts. The variants in virus libraries are sorted by thedecreasing order of standard score and their scores from respective DNAlibraries are mapped onto them.

FIG. 47 shows correlations between the standard scores of read countsfor the DNA and virus libraries (n=1 per library) produced by thesynthetic pool and PCR pool methods is determined by linearleast-squares regression, and the regression line (best fit) and R2representing the coefficient of determination.

FIG. 48 shows distributions of capsid libraries from brain tissue of twomice used in each Cre line selection, as produced by the synthetic pool(left) and PCR pool (right) designs. The distribution of spike-inlibrary introduced in the synthetic pool library design is shown atcenter.

FIG. 49 illustrates correlations of enrichment scores of variants fromthe brain libraries (n=2 per Cre line, mean is plotted) produced bysynthetic pool and PCR pool methods is determined by the same methoddescribed with respect to FIG. 47.

FIG. 50 shows correlation analysis between the enrichment score (log10)of two alternate codon replicates of variants from the GFAP-Cre (left),SNAP-Cre (center), and Syn-Cre (right) brain libraries by linearleast-squares regression (n=2 per Cre line, mean is plotted). The dottedline separates the high-confidence signal from noise. High confidencesignal (below) is assessed by a linear regression line (best fit) and R2represents the coefficient of determination.

FIG. 51 shows the difference in enrichment score between the two codonreplicates of a variant, across different brain libraries, with over8000 variants recovered in replicates.

FIG. 52 shows heatmaps represent the magnitude (log2 fold change) of AAbias in “output” library 1 normalized to “input” library 2 that reachstatistical significance (boxed if P-value <0.0001, two-sided,two-proportion z-test, except in R1 DNA normalized to known NNK templatewhere one-proportion z-test was performed, and P-values corrected formultiple comparisons using Bonferroni correction). R1 DNA librarynormalized to NNK template (top left, ˜9 million sequences), R1 virusnormalized to R1 DNA libraries (bottom left, ˜10 million sequences), R2GFAP library with enrichment score above 1.0 in brain normalized to R2virus (top right, 20 sequences,) and R2 SNAP library with enrichmentscore above 1.2 normalized to R2 virus (bottom right, 17 sequences) areshown (n=1 for DNA, virus, and n=2 for brain libraries).

FIG. 53 shows clustering analysis of positively enriched variants fromTek, GFAP, and combined neuron brain libraries (SNAP and Syn) by PCRpool design, and by synthetic pool design with spike-in library areshown with size of nodes representing their relative enrichment inbrain, and the thickness of edges (connecting lines) representing theextent of shared AA identity between nodes. A distinct family ishighlighted in yellow with the corresponding AA frequency logo below (AAsize reflects prevalence and color coded based on AA properties).

FIG. 54 shows expression of AAV9 (top row) and AAV-PHP.V1 (bottom row)packaging CAG promoter driving mNeonGreen across all organs (n=3, 3×10¹¹vg dose per adult C57BL/6J mouse, 3 weeks of expression).

FIG. 55 shows expression in cortical astrocytes (S100+) after IVdelivery of AAV-PHP.V1 (left) and AAV-PHP.eB (right) capsids carryingthe GfABC1D promoter driving expression of nuclear-localized mTurquoise2reporter (1×10¹² vg dose per adult mouse, 4 weeks of expression).Percentage of cortical S100+cells that overlapped with mTurquoise2expression is quantified (n=2, each data point is mean from 3 images permouse).

FIG. 56. shows expression of single-stranded (ss) AAV9, PHP.eB inAi14-tdTomato reporter adult mouse (n=2-3 per group, 3×10¹¹ vg dose peradult mouse, 3 weeks of expression)

FIG. 57 shows expression of PHP.V1 in Ai14-tdTomato reporter adult mouse(n=2-3 per group, 3×10¹¹ vg dose per adult mouse, 3 weeks of expression)

FIG. 58 shows expression of packaging Ple261 promoter carrying iCretransgene in Ai14-tdTomato reporter adult mouse (n=2-3 per group, 3×10¹¹vg dose per adult mouse, 3 weeks of expression) on the left. In theright column, FIG. 58 shows expression of self-complementary (sc)AAV-PHP.V1 carrying CB6 ubiquitous promoter driving EGFP (above) and CAGpromoter driving EGFP (below). Lectin DyLight 594 staining is also shown(n=2-3, 3×10¹¹ vg dose per adult C57BL/6J mouse, 2 weeks of expression).Experiments in FIGS. 56, 57, and the left column of FIG. 58 are reportedfrom one independent trial from a fresh batch of viruses, and titered inthe same assay for dosage consistency. Experiments in FIG. 58 werevalidated in two independent trials (n=2 per group).

FIG. 59 shows transduction of mouse brain by the AAV-PHP.V2 variant andcontrol AAV9, carrying the CAG promoter that drives the expression ofmNeonGreen (n=3, 3×10¹¹ vg IV dose per C57BL/6J adult mouse, 3 weeks ofexpression). The sagittal brain images (left) were acquired using thesame microscope settings to that of the sagittal brain images in FIG.14.

Higher magnification images of AAV-PHP.V2 transduced brain sectionsstained with aGLUT or aS100 or aOlig2 are shown.

FIG. 60 shows Transduction of brain vasculature by AAV-PHP.V2 carryingCAG-DIO-EYFP in Tek-Cre adult mice (left, 1×10¹² vg IV dose per mouse, 4weeks of expression), and its efficiency (right) is determined by theoverlap of aGLUT1 staining with EYFP expression across different brainareas (n=2, mean of 3 images per brain region per mouse).

FIG. 61 shows transduction of astrocytes by AAV-PHP.V2 in GFAP-Cre adultmouse (1×10¹² vg IV dose per mouse, 4 weeks of expression). Percentageof cortical S100⁺cells that overlapped with EYFP expression isquantified (n=2, mean of 3 images per mouse).

FIG. 62 shows transduction levels of liver hepatocytes quantified as thepercentage of DAPI⁺cells that are EGFP⁺(n=3, vectors packaged withCAG-2xNLS-EGFP, lx10¹¹ vg IV dose/adult C57BL/6J mouse, 3 weeks ofexpression, mean±S.E.M, 4 images per mouse per group. One-way ANOVA,non-parametric Kruskal-Wallis test gave an approximate P-value of0.0088).

FIG. 63 shows transduction of brain tissue by AAV-PHP.B4, B7, AAV-PHP.X1(ARQMDLS), and AAV-PHP.X2 (TNKVGNI) packaging CAG-mNeonGreen genome(n=3, lx10¹¹ vg IV dose/adult C57BL/6J mouse, 3 weeks of expression),were acquired using the same microscope settings to that of AAV9 andAAV-PHP.V1 sagittal brain images in FIG. 14.

FIG. 64 shows transduction of the brain by AAV-PHP.B8 using theCAG-mRuby2 genome (n=3, 3×10¹¹ vg IV dose/ adult C57BL/6J mouse, 3 weeksof expression).

FIG. 65 shows Transduction of AAV9 (left), AAV-PHP.X3 (QNVTKGV) (middle)and AAV-PHP.X4 (LNAIKNI) (right) vectors packaging CAG-2xNLS-EGFP (n=2,1×10¹¹ vg IV dose/ adult C57BL/6J mouse, 3 weeks of expression). Data inFIGS. 62-65 is reported from one independent trial.

FIGS. 66-67 show distributions of R1 (FIGS. 66) and R2 (FIG. 67) brainlibraries (at AA level, standard score (SS) of RCs sorted in decreasingorder of scores). The SS for AAV-PHP.N and AAV-PHP.eB across librariesare mapped on the zoomed-in view of this plot (dotted line box).

FIG. 68 shows a heatmap of AA distributions across the diversifiedregion of the positively enriched variants from R2 liver library (top100 sequences) normalized to the R2 virus (input library).

FIG. 69 shows clustering analysis of positively enriched variants fromGFAP and Vglut2 brain libraries are shown with size of nodesrepresenting their relative negative enrichment in liver, and thethickness of edges (connecting lines) representing their relativeidentity between nodes.

FIG. 70 shows expression of AAV-PHP.B (top row) and AAV-PHP.N (bottomrow) packaged with ssAAV:CAG-mNeonGreen across all organs (n=3, 3×10¹¹vg IV dose per adult C57BL/6J mouse, 3 weeks of expression).

FIG. 71 shows transduction of mouse brain by the AAV-PHP.N variant,carrying the CAG promoter that drives the expression of mNeonGreen (n=3,1×10¹¹ vg IV dose per C57BL/6J adult mouse, 3 weeks of expression).Fluorescence in situ hybridization chain reaction (FITC-HCR) was used tolabel excitatory neurons with Vglutl and inhibitory neurons with Gadl.Few cells where EGFP expression co-localized with specific cell markersare highlighted by asterisks symbol.

FIG. 72 shows transduction of AAV9, AAV-PHP.eB and AAV-PHP.V1 in humanbrain microvascular endothelial cell culture (HBMEC). The vectors werepackaged with ssAAV:CAG-mNeongreen. The mean fluorescence intensityacross the groups were quantified (n=3 tissue culture wells of 0.95 cm²surface area per group, 3 images per well per group per dose was imagedafter three days of expression, doses lx10⁸ vg and lx10¹⁰ vg per 0.95cm² surface area). A two-way ANOVA with correction for multiplecomparisons using Tukey's test gave adjusted P-value of 0.0051 for AAV9vs PHP.V1, 0.0096 for PHP.eB vs PHP.V1, 0.8222 for AAV9 vs PHP.eB forlx10⁸ vg, and 0.0052 for AAV9 vs PHP.V1, 0.0049 for PHP.eB vs PHP.V1,0.9996 for AAV9 vs PHP.eB for 1×10¹° vg (**P <0.01, is shown and P >0.05is not shown on the plot; mean ±S.E.M., 95% CI).

FIG. 73 shows the transduction of cortex brain region by AAV-PHP.B,AAV-PHP.C2 and AAV-PHP.C3 across two different mouse strains: C57BL/6Jand BALB/cJ. The vectors were packaged with ssAAV:CAG-mNeongreen (n=2-3per group, lx10¹¹ vg IV dose/adult mouse, 3 weeks of expression). Allimages were acquired using the same microscope settings. The datareported in FIGS. 72 and 73 are from one independent trial where allviruses were freshly prepared and tittered in the same assay for dosageconsistency, with additional validation for AAV-PHP.C2 and AAV-PHP.C3 inan independent trial for BALB/cJ.

FIG. 74 provides details of a spike-in library of AAV9 and ˜50additional variants (and their alternative codon duplicates), identifiedin prior publications (includes well characterized variants likeAAV-PHP.B or AAVPHP.eB as well as many variants identified using theprevious methodology but uncharacterized in vivo) act as internalselection controls and standards for the relative performance of the newvariants. Deverman, B. E. et al. Cre-dependent selection yields AAVvariants for widespread gene transfer to the adult brain. Nat.Biotechnol. 34,204-209 (2016) and Chan, K. Y. et al. Engineered AAVs forefficient noninvasive gene delivery to the central and peripheralnervous systems. Nat. Neurosci. 20, 1172-1179 (2017); the content ofeach of which is incorporated herein by reference. The spike-in librarywas generated as part of the synthetic pool library.

FIG. 75 shows enrichment scores for the spike-in library.

DETAILED DESCRIPTION

While preferred instances of the present disclosure have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch instances are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the disclosure. It should be understood thatvarious alternatives to the instances of the disclosure described hereinmay be employed in practicing the disclosure. It is intended that thefollowing claims define the scope of the disclosure and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

Provided herein are modified adeno-associated (AAV) virus capsidcompositions useful for integrating a transgene into a target cell orenvironment (e.g., a cell-type or tissue) in a subject when they areadministered systemically (e.g., intravenous, intranasal) to thesubject. The modified AAV capsid proteins of the present disclosurecomprise at least one insertion or substitution of an amino acid in acorresponding parental AAV capsid protein that confers a desired tropismsuch as an increased or decreased specificity as compared to a referenceAAV capsid protein, or increased or decreased transgene transductionefficiency as compared to a reference AAV capsid protein.

Disclosed herein are AAV capsids engineered with desired tropisms, suchas an increased specificity of viral transduction in a target in vivoenvironment, such as a tissue or cell. In some embodiments, the AAVcapsids of the present disclosure are engineered to specifically targetthe central nervous system (CNS) of a subject. In some embodiments, theAAV capsids of the present disclosure are engineered to specificallytarget the liver of a subject. The AAV capsids can encapsidate a viralvector with a heterologous nucleic acid encoding, for example, atherapeutic gene expression product. Highly specific transduction of theheterologous nucleic acid in a target in vivo environment (e.g., brain,liver) can be achieved upon systemic delivery to a subject of the AAVcapsid of the present disclosure encapsidating a heterologous nucleicacid. The AAV capsids disclosed herein are advantageous for manyapplications, such as diagnosing and/or treating monogenetic disordersof the brain (e.g., GLUT1-deficiency syndrome, mucoploysaccharidosistype IIIC), producing adoptive cellular therapies, and biomedicalresearch applications.

The AAV capsids comprise AAV capsid proteins (e.g., VP1, VP2, and VP3),each with an insertion or a substitution of at least one amino acid atan amino acid in the 588 loop of a parental AAV capsid protein structure(AAV9 VP1 numbering). The 588 loop contains the site of heparan sulfatebinding of AAV2 and amenable to peptide display. The only knownreceptors for AAV9 is N-linked terminal galactose and AAV receptor(AAVR), but many indications point toward there being others.Modifications to AAV9 588 loop are shown herein to confer an increasedspecificity and transgene transduction in target in vivo environments ascompared to a reference AAV in rodent models. In some cases, theparental AAV capsid protein has an AAV serotype 9 (alias AAV9).

The most common method of AAV-mediated transgene delivery is by directinjection to the target in vivo environment, which is disadvantageousfor many reasons, including risk of injury or death, pain, and highercost, as compared to less invasive methods. For example, intracranialinjection can cause hemorrhaging of the brain. Previous AAV-mediateddelivery by intravenous administration avoids a need for a directinjection, but suffers from reduced specificity for the target in vivoenvironment (e.g., tissue or cell) resulting in off-target transductionevents and necessitating a larger viral load to achieve sufficienttherapeutic levels in the target in vivo environment. This is especiallyevident when the AAV must cross the blood brain barrier (BBB).

Methods are disclosed comprising systemically administering an AAVcapsid of the present disclosure encapsidating a viral vector comprisinga transgene (e.g., therapeutic nucleic acid) with an increasedspecificity, as compared to a reference AAV capsid protein. The AAVcapsids of the present disclosure are capable of crossing the BBB, andtransducing a transgene in a particular target cell-type (e.g., neuron,endothelial cell) in a subject. Accordingly, the AAV capsid proteins ofthe present disclosure are suitable for transgene therapy to treat humandisease, particularly disease that effects the target in vivoenvironment.

The transgenes contained in a recombinant AAV (rAAV) vector andencapsidated by the AAV capsid proteins of the present disclosure arealso provided herein. The transgenes disclosed herein are delivered to asubject for a variety of purposes, such as to treat a disease orcondition in the subject. The transgene can be gene editing componentsthat modulate the activity or expression of a target gene or geneexpression product. Alternatively, the transgene is a gene encoding atherapeutic gene expression product that is effective to modulate theactivity or expression of itself, or another target gene or geneexpression product.

Provided herein, are methods of identifying the 7-mer or 3-mer peptideinsertions comprising multiplexed Cre recombination-based AAV targetedevolution (M-CREATE). The M-CREATE method of the present disclosuresupports (1) the calculation of a true enrichment score for each variantby using deep sequencing to correct for biases in viral production priorto selection, (2) reduced propagation of bias in successive rounds ofselection through the creation of a post-round 1 synthetic pool librarywith equal variant representation, (3) the reduction of false positivesby including two codon replicates of each selected variant in the pool,and (4) both positive and negative selection criteria by comparing deepsequencing of recovered capsid libraries among multiple targets (cellstypes or organs). These features allow informed, confident choices onvariants worthy of validation and characterization in vivo.

Disclosed herein are methods of producing the AAV capsids comprising theAAV capsid proteins and viral vector encoding a therapeutic nucleicacid. The AAV capsid proteins are produced by introducing into a cell(e.g., immortalized stem cell) a first vector encoding the transgene(e.g., containing the therapeutic nucleic acid), a second vectorencoding the AAV genome with a AAV capsid protein, and a third vectorencoding helper virus proteins, required for assembly of the AAV capsidstructure and packaging of the transgene in the AAV capsid. Theassembled AAV capsid can be isolated and purified from the cell usingsuitable methods known in the art.

The recombinant AAV vectors comprising a nucleic acid sequence encodingthe AAV capsid proteins of the present disclosure as also providedherein. For example, the viral vectors of the present disclosurecomprise a nucleic acid sequence comprising the AAV viral Cap (Capsid)encoding VP1, VP2, and VP3, at least one of which is modified to producethe AAV capsid proteins of the present disclosure. The recombinant AAVvector provided can be derived from an AAV serotype (e.g., AAV9).

I. COMPOSITIONS

Recombinant adeno-associated virus (rAAV) mediated gene deliveryleverages the AAV mechanism of viral transduction for nuclear expressionof an episomal heterologous nucleic acid (e.g., a transgene, therapeuticnucleic acid). Upon delivery to a host in vivo environment, a rAAV will(1) bind or attach to cellular surface receptors on the target cell, (2)endocytose, (3) traffic to the nucleus, (4) uncoat the virus to releasethe encapsidated heterologous nucleic acid , (5) convert of theheterologous nucleic acid from single-stranded to double-stranded DNA asa template for transcription in the nucleus, and (6) transcribe of theepisomal heterologous nucleic acid in the nucleus of the host cell(“transduction”). rAAVs engineered to have an increased specificity(binding to cellular surface receptors on the target cell) andtransduction efficiency (transcription of the episomal heterologousnucleic acid in the host cell) are desirable for gene therapyapplications.

A rAAV comprises an AAV capsid that can be engineered to encapsidate aheterologous nucleic acid (e.g., therapeutic nucleic acid, gene editingmachinery). The AAV capsid is made up of three AAV capsid proteinmonomers, VP1, VP2, and VP3. Sixty copies of these three VP proteinsinteract in a 1:1:10 ratio to form the viral capsid (FIG. 2). VP1 coversthe whole of VP2 protein in addition to a ˜137 amino acid N-terminalregion (VP1u), VP2 covers the whole of VP3 in addition to ˜65 amino acidN-terminal region (VP1/2 common region). The three capsid proteins sharea conserved amino acid sequence of VP3, which in some cases is theregion beginning at amino acid position 138 (e.g., AA139-736).

The AAV VP3 structure contains highly conserved regions that are commonto all serotypes, a core eight-stranded β-barrel motif (f3B-(3I) and asmall a-helix (aA). The loop regions inserted between the β-strandsconsist of the distinctive HI loop between β-strands H and I, the DEloop between β-strands D and E, and nine variable regions (VRs), whichform the top of the loops. These VRs, such as the AA588 loop, are foundon the capsid surface and can be associated with specific functionalroles in the AAV life cycle including receptor binding, transduction andantigenic specificity.

Disclosed herein are AAV capsids comprising AAV capsid proteins with asubstitution at the 588 loop that confer a desired tropism characterizedby a higher specificity for transduction in specific cell-types,including for e.g., brain cell types (e.g., brain endothelial cells,neurons, astrocytes) and liver cell types. In particular, the AAV capsidproteins disclosed herein enable rAAV-mediated transduction of aheterologous nucleic acid (e.g., transgene) in the brain or the liver ofa subject. The AAV capsids of the present disclosure, or the AAV capsidproteins, may be formulated as a pharmaceutical composition. Inaddition, the AAV capsids or the AAV capsid proteins can be isolated andpurified to be used for a variety of applications.

A. Adeno-Associated Virus (AAV) Capsid Proteins

Disclosed herein are recombinant AAV (rAAV) capsids comprise AAV capsidproteins that are engineered with a modified capsid protein (e.g., VP1,VP2, VP3). In some embodiments, the rAAV capsid proteins of the presentdisclosure are generated using the methods disclosed herein (e.g.,M-CREATE). In some embodiments, the AAV capsid proteins are used in themethods of delivering a therapeutic nucleic acid (e.g., a transgene) toa subject. In some instances, the rAAV capsid proteins have desired AAVtropisms rendering them particularly suitable for certain therapeuticapplications, e.g., the treatment of a disease or disorder in a subjectsuch as those disclosed herein.

The rAAV capsid proteins are engineered for optimized entry into andthrough the blood brain barrier (BBB) of a subject upon systemicadministration of the rAAV to the subject, such as those provided inTables 2-3, and FIG. 33. Prior methods of AAV-mediated delivery of atherapeutic transgene to the brain required intracranial injection.Intracranial injection is an invasive procedure that causes a subjectdiscomfort, and in some cases, pain. For example, intracranial injectioncan cause hemorrhaging of the brain. Additionally, intracranial deliveryhas limited spread and is highly heterogeneous. The rAAV capsid proteinsprovided in Tables 2-4, and FIG. 33 are engineered to have tropisms thateliminate the need for intracranial injection, while also achievingwidespread and efficient transduction of an encapsidated transgene. Inparticular, the tropisms comprise at least one of an increasedspecificity and efficiency (e.g., of viral transduction) in the centralnervous system (CNS) of a subject, as compared to a reference AAV.

The engineered AAV capsid proteins described herein have, in some cases,an insertion of an amino acid that is heterologous to the parental AAVcapsid protein at the amino acid position in the 588 loop. In someembodiments, the amino acid is not endogenous to the parental AAV capsidprotein at the amino acid position of the insertion. The amino acid maybe a naturally occurring amino acid in the same or equivalent amino acidposition as the insertion of the substitution in a different AAV capsidprotein.

Aspects provided herein provide amino acid insertions comprising sevenamino acid polymer (7-mer) inserted at AA588 589, and may additionallyinclude a substitution of one or two amino acids at amino acid positionsflanking the 7-mer sequence (e.g., AA587-588 and/or AA589-590) toproduce an eleven amino acid polymer (11-mer) at the 588 loop of aparental AAV capsid protein. The 7-mers described herein wereadvantageously generated using polymerase chain reaction (PCR) withdegenerate primers, where each of the seven amino acids is encoded by adeoxyribose nucleic acid (DNA) sequence N-N-K. “N” is any of the fourDNA nucleotides and K is guanine (G) or thymine (T). This method ofgenerating random 7-mer amino acid sequences enables 1.28 billionpossible combinations at the protein level. Since the 7-mers developedare random, some amino acids in the 7-mer may be naturally occurring inthe AAC capsid protein at that amino acid position, while other aminoacids may differ.

Recombinant AAVs (rAAVs) were generated, each with a unique 7-mer or11-mer at the 588 loop and each encapsidating a reporter gene that, whenadministered systemically in multiple transgenic animals, enabled theselective amplification and recovery of sequences that effectivelytransduced the reporter gene in a target in vivo environment of thetransgenic animal. 7-mers and 11-mers that were found to be positivelyenriched in the target in vivo environment (e.g, central nervous system,liver) are provided herein. “Enrichment” is the prevalence of a given7-mer or an 11-mer in the tissue of the in vivo environment compared toits prevalence in the viral library that was administered to thetransgenic animal. An enrichment score above 0 indicates a positiveenrichment. An enrichment score below 0 indicates a negative enrichment.A subset of the rAAVs with desired enrichment profiles were testedindividually in vivo to determine exact systemic expression (e.g.,specificity and transduction efficiency). rAAVs from this subsetexhibiting a desired tropism comprising increased specificity of viraltransduction, and in some cases, transduction efficiency are consideredto be uniquely suited for targeted rAAV-mediated transgene deliveryuseful for a wide variety of purposes (e.g., therapeutic, diagnostic,scientific discovery).

The rAAV particles with the 7-mers or 11-mers described herein have anincreased transduction efficiency in a target in vivo environment (e.g.,tissue or cell type). In some instances, the increased transductionefficiency comprises a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold,23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold,31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold,39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold,47-fold, 48-fold, 49-fold, 50-fold, 75-fold, or 100-fold increase, ormore, relative to a reference AAV. In some instances, the increasedtransduction efficiency is at least 30-fold. In some instances, theincreased transduction efficiency is at least 40-fold. In someinstances, the increased transduction efficiency is at least 50-fold. Insome instances, the increased transduction efficiency is at least60-fold. In some instances, the increased transduction efficiency is atleast 80-fold. In some instances, the increased transduction efficiencyis at least 90-fold. In some instances, the increased transductionefficiency is at least 100-fold.

The rAAV particles with the 7-mers or 11-mers described herein have anincreased specificity in a target in vivo environment (e.g., tissue orcell type), as compared to a reference AAV. Detecting whether a rAAVpossesses more or less specificity for a target in vivo environment thana reference AAV, includes measuring a level of gene expression product(e.g., RNA or protein) expressed from the heterologous nucleic acidencapsidated by the rAAV in a tissue sample obtained from the target invivo environment in a subject; and comparing the measured level to acontrol level (such as, for e.g., the gene expression product expressedfrom a heterologous nucleic acid encapsidated by a reference AAV (e.g.,AAV9)). Suitable methods for measuring expression of a gene expressionproduct luciferase reporter assay and quantitative polymerase chainreaction (qPCR).

The increased specificity is correlated with an increased enrichment inthe target in vivo environment, which in some cases is represented withan enrichment score provided herein in FIGS. 33-35. As a non-limitingexample, AAV-PHP.V2 (TTLKPFL), which is shown herein to be positivelyenriched in the brain (enrichment score of ˜2.51) also exhibited anincrease in reporter gene expression (e.g., measured by fluorescencereporter assay) in the brain (-60% of cortical brain vasculature cellsand ˜60% cortical astrocytes transduced with the reporter gene) ascompared to a reference AAV9 (˜0%). Without being bound by a particulartheory, the inventors of the present disclosure would expect to see thiscorrelation for all rAAVs disclosed herein, and further, would expectthat a more significant the enrichment score (whether negative orpositive) would correlate with a more significant specificity to the invivo environment(s) as indicated by a measured level of the geneexpression product in the in vivo environment(s).

Transduction efficiency, as disclosed herein, may be measured by atleast one of (1) a number of cells in a target in vivo or off-target invivo environment expressing the heterologous nucleic acid encapsidatedby the modified AAV capsid proteins disclosed herein, and (2) a quantityof expression of the heterologous nucleic acid in a single cell.Specificity for a target in vivo environment may be inferred when apresence, or an increase in a level, of rAAV-mediated transduction in atarget in vivo environment is observed, as compared to a reference AAV.A lack of, or reduced, specificity to an off-target in vivo environmentmay be inferred when an absence, or a decrease in a level, ofrAAV-mediated transduction in the off-target in vivo environment isobserved, as compared to a reference AAV.

The reference AAV may have a serotype selected from the group consistingof AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, or variant thereof. For example, the reference AAV can have aserotype selected from the group consisting of AAV-PHP.B, AAV-PHP.eB,and AAV-PHP.S.

The rAAV capsid proteins of the present disclosure comprise an insertionof an amino acid in an amino acid sequence of an AAV capsid protein. TheAAV capsid, from which an engineered AAV capsid protein of the presentdisclosure is produced, is referred to as a “parental” AAV capsid. Insome cases, the parental AAV has a serotype selected from the groupconsisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, and AAV12. The complete genome of AAV-1 is provided inGenBank Accession No. NC 002077; the complete genome of AAV-2 isprovided in GenBank Accession No. NC 001401 and Srivastava et al., J.Virol., 45: 555-564 (1983); the complete genome of AAV-3 is provided inGenBank Accession No. NC 1829; the complete genome of AAV-4 is providedin GenBank Accession No. NC 001829; the AAV-5 genome is provided inGenBank Accession No. AF085716; the complete genome of AAV-6 is providedin GenBank Accession No. NC 00 1862; at least portions of AAV-7 andAAV-8 genomes are provided in GenBank Accession Nos. AX753246 andAX753249, respectively; the AAV -9 genome is provided in Gao et al., J.Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol.Ther., 13(1): 67-76 (2006); the AAV-11 genome is provided in Virology,330(2): 375-383 (2004); portions of the AAV-12 genome are provided inGenbank Accession No. DQ813647; portions of the AAV-13 genome areprovided in Genbank Accession No. EU285562.

In some cases, the parental AAV is derived from an AAV with a serotypeselected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The AAV capsid proteinthat is “derived” from another may be a variant AAV capsid protein. Avariant may include, for example, a heterologous amino acid in an aminoacid sequence of the AAV capsid protein. The heterologous amino acid maybe non-naturally occurring in the AAV capsid protein. The heterologousamino acid may be naturally occurring in a different AAV capsid protein.In some instances, the parental AAV capsid is described in U.S. Pat.App. Ser. Nos. 6²/₇36,904; 16/582,635; 62/832,812; and 62/832,826; thecontent of each of which is incorporated herein. For example, theparental AAV capsid may be modified at the 455 loop of the AAV capsidprotein (e.g., substitutions of 7-mer at AA452-458, AAV9 VP1 numbering).

In some instances, the parental AAV is AAV9. In some instances, theamino acid sequence of the AAV9 capsid protein comprises SEQ ID NO: 1.The amino acid sequence of AAV9 VP1 capsid protein (>trIQ6JC401Q6JC409VIRU Capsid protein VP1 OS=Adeno-associated virus 9 OX=235455 GN=capPE=1 SV=1) is provided in SEQ ID NO: 1(MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPS GVGSLTMAS GGGAPVADNNEGADGVGSSS GNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTS GGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS QAVGRSSFYCLEYFPS QMLRTGNNFQFSYEFENVPFHSSYAHS QSLDRLMNPLIDQYLYYLSKTINGS GQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLS GSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL). In some instances, the parentalAAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 1.

Disclosed herein are insertions of an amino acid (or amino acidsequence) in an amino acid sequence of an AAV capsid protein at an aminoacid position between amino acid 588 and amino acid 589. As used herein,“AA588_589” indicates that the insertion of the amino acid (or aminoacid sequence) is immediately after an amino acid (AA) at position 588and immediately before an AA at position 589 within an amino acidsequence of a parental AAV VP capsid protein (VP1 numbering). Aminoacids 587-591 include a motif comprising “AQAQA” as set forth in SEQ IDNO: 1. Exemplary AAV capsid protein sequences are provided in Table 1.For example, QAVRTSL is inserted at AA588_589 in an AAV9 capsid aminoacid sequence, and is provided SEQ ID NO: 3. In another example, TLAVPFKis inserted at AA588_589 in an AAV9 capsid amino acid sequence, and isprovided in SEQ ID NO: 2. It is envisioned that the 7-mer insertionsdisclosed herein (FIGS. 33-35, Tables 2-4) may be inserted at AA588_589in an amino acid sequence of a parental AAV9 capsid protein, a variantthereof, or equivalent amino acid position a parental AAV of a differentserotype (e.g., AAV1, AAV2, AAV3, and the like).

The 11-mers described herein may, in some cases, comprise a 7-merinsertion at AA588 589, and substitutions of one or more amino acids atamino acid positions AA587-590. In some cases, the amino acids 587-590are substituted with an amino acid that is not endogenous to theparental AAV capsid protein at that position. In some cases, AA587 issubstituted with D (e.g., A587D). In some cases, AA587 is substitutedwith an A (e.g., Q587A). In some cases, AA587 is substituted with an S(e.g., Q587S). In some cases, AA587 is substituted with a G (e.g.,Q587G). In some cases, AA588 is substituted with a G (e.g., Q588G). Insome cases, AA589 is substituted with an N (e.g., A589N). In some cases,AA590 is substituted with a P (e.g., A590). In a non-limiting exampleSEQ ID NO: 4 (PHP-AAV.eB) comprises an insertion at AA588_AA589 ofTLAVPFK, and substitutions A587D and Q588G. In another non-limitingexample, SEQ ID NO: 5 (PHP-AAV.N) comprises an insertion at AA588_AA589of TTLKPFS, and substitutions A587D, Q588G, A589N, and Q590P. It isenvisioned that any 7-mer insertion disclosed herein in addition to asubstitution with any amino acid at amino acid positions 587-590 maycomprise an 11-mer.

TABLE 1 Exemplary AAV Capsid Protein Sequences SEQ ID NO: IdentifierSequence 2 AAV- MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN PHP.BARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTLAVPFKAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV EFAVNTEGVYSEPRPIGTRYLTRNL 3AAV- MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN PHP.SARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQQAVRTSLAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV EFAVNTEGVYSEPRPIGTRYLTRNL 4AAV- MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN PHP.eBARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSDGTLAVPFKAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV EFAVNTEGVYSEPRPIGTRYLTRNL 5AAV- MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN PHP.NARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTLAVPFSNPAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV EFAVNTEGVYSEPRPIGTRYLTRNL

The rAAV capsid proteins described herein may be isolated and purified.The AAV may be isolated and purified by methods standard in the art suchas by column chromatography or cesium chloride gradients. Methods forpurifying AAV from helper virus are known in the art and may includemethods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427-443(2002); U.S. Patent No. 6,566,118 and WO 98/09657.

The rAAV capsid protein can be conjugated to a nanoparticle, a secondmolecule, or a viral capsid protein. In some cases, the nanoparticle orviral capsid protein would encapsidate the therapeutic nucleic aciddescribed herein. In some instances, the second molecule is atherapeutic agent, e.g., a small molecule, antibody, antigen-bindingfragment, peptide, or protein, such as those described herein. In someinstances, the second molecule is a detectable moiety. For example, themodified AAV capsid protein conjugated to a detectable moiety may beused for in vitro, ex vivo, or in vivo biomedical research applications,the detectable moiety used to visualize the modified capsid protein. Themodified AAV capsid protein conjugated to a detectable moiety may alsobe used for diagnostic purposes.

AAV Capsid Proteins Targeting the Central Nervous System

Disclosed herein are AAV capsid proteins with a substitution or aninsertion of at least one amino acid at an amino acid position describedabove in a parental AAV capsid protein that confers an increasedspecificity for a central nervous system (CNS) or peripheral nervoussystem (PNS) in a subject, even when delivered systemically. One of themany advantages of the AAV capsid proteins described herein is theirability to target the CNS and penetrate the blood brain barrier (BBB).

The in vivo environment can be a cell. The cell can be a cell-typeselected from the group consisting of a central nervous system (CNS)cell and a peripheral nervous system (PNS) cell. Non-limiting examplesof CNS cells include a neuron and a glial cell. Glial cells can beselected from the group consisting of an oligodendrocyte, an ependymalcell, and an astrocyte. Non-limiting examples of a PNS cell includes aneuron or a glial cell. The glial cell can be selected from the groupconsisting of a Schwann cell, a satellite cell, and an enteric glialcell.

The in vivo environment can be a tissue. The tissue can be the brain, orthe spinal cord. The tissue can be a region of an organ, example, thecerebrum, the cerebellum, the brainstem, the cortex, the striatum, thethalamus, the lateral ventricles, the putamen, the hypothalamus, themedulla, the pons, the hippocampus, the amygdala, the motor cortex, or acombination thereof.

Disclosed herein are AAV capsid proteins with at least one amino acidinsertion or substitution in a parental AAV capsid protein. Theinsertion or substitution can be of at least one, two, three, four,five, six, seven, eight, nine, ten, or eleven amino acids, or more. Insome instances, the amino acids are contiguous. In some instances, theamino acids are not contiguous.

In some instances, the insertion is of at least one amino acid isprovided in any one of the sequences provided in any one of Tables 2-3,or FIG. 6. In some instances, the insertion is of at least two aminoacids provided in any one of the sequences provided in any one of Tables2-3, or FIG. 6. In some instances, the insertion is of at least threeamino acids provided in any one of the sequences provided in any one ofTables 2-3, or FIG. 6. In some instances, the insertion is of at leastfour amino acids provided in any one of the sequences provided in anyone of Tables 2-3, or FIG. 6. In some instances, the insertion is of atleast five amino acids provided in any one of Tables 2-3, or FIG. 6. Insome instances, the insertion is of at least six amino acids provided inany one of Tables 2-3, or FIG. 6. In some instances, the insertion is ofat least seven amino acids provided in any one of Tables 2-3, or FIG. 6.

Disclosed herein are AAV capsid proteins with an insertion of at leastone amino acid X1, wherein X1 is selected from the group consisting ofA, E, D, G, R, S and T. In some instances, the insertion furthercomprises two amino acids, wherein X2 is selected from the groupconsisting of A, G, I, L, M, N, Q, R, T, and Y. In some instances, theinsertion further comprises three amino acids, wherein X3 is selectedfrom the group consisting of E, K, L, T, and Q. In some instances, theinsertion further comprises at least four amino acids, wherein X1 isselected from the group consisting of A, E, D, G, R, S and T, X2 isselected from the group consisting of A, G, I, L, M, N, Q, R, T, and Y,X3 is selected from the group consisting of E, K, L, T, and Q, and X4 isselected from the group consisting of G, I, K, L, M, R, T, and V. Insome instances, the insertion further comprises five amino acids whereinX5 is selected from the group consisting of A, D, G, P, L, Q, and V. Insome instances, the insertion further comprises at least six aminoacids, wherein X6 is selected from the group consisting of F, K, L, N,P, Q, S, and V. In some instances, the insertion further comprises atleast seven amino acids, wherein X7 is selected from the groupconsisting of I, K, L, P, S, and V.

In some embodiments, Xl, X2, X3, X4, X5, X6, and X7 are contiguous(X1-X2-X3-X4-X5-X6-X7). In some embodiments, any two of Xl, X2, X3, X4,X5, X6, and X7 are contiguous. In some embodiments, any three of Xl, X2,X3, X4, X5, X6, and X7 are contiguous. In some embodiments, any four ofXl, X2, X3, X4, X5, X6, and X7 are contiguous. In some embodiments, anyfive of Xl, X2, X3, X4, X5, X6, and X7 are contiguous. In someembodiments, any six of Xl, X2, X3, X4, X5, X6, and X7 are contiguous.In some embodiments, any seven of Xl, X2, X3, X4, X5, X6, and X7 arecontiguous. In some embodiments, Xl, X2, X3, X4, X5, X6, and X7 are notcontiguous. In some embodiments, the insertion is not TLAVPFK.

The 7-mers disclosed herein, in some cases share a common motif. A 7-mer(X1-X2-X3-X4-X5-X6-X7) may in some cases advantageously have a T inposition Xl, an L in position X2, a P in positive X5, an F in positionX6, and a K or L in position X7. In some embodiments, the 7-mercomprises T-L-X3-X4-P-F-K, wherein X3 and X4 are any amino acid. In someembodiments, the 7-mer comprises T-L-X3-X4-P-F-L, wherein X3 and X4 areany amino acid. In some cases, X3 is not an A. In some cases, X3 is A,S, Q, or E or L. In some cases, X4 is not a V. In some cases, X4 is R,K, V, or a Q.

In some cases, the 7-mer may be X1 L A V PF K, wherein X1 is any aminoacid other than T, S, or N; X1-X2-A-V-P-F-K, wherein X1 is any aminoacid other than T, S, or N, and X2 is any amino acid other than L or V;or Xl-X2-X3-V-P-F-K, wherein X1 is any amino acid other than T, S, or N,X2 is any amino acid other than L or V, and X3 is any amino acid otherthan A, S, Q, P, or T; or Xl-X2-X3-X4-P-F-K, wherein X1 is any aminoacid other than T, S, or N, X2 is any amino acid other than L or V, X3is any amino acid other than A, S, Q, P, or T, and X4 is any amino acidother than V, T, Q, N, L, or M. In some instances, the 7-mer isT-L-A-X4-P-F-K, wherein X is any amino acid other than V. In someinstances, the 7-mer is T-L-A-X4-P-F-K, wherein X is any amino acidother than, T, Q, N, L, or M.

In some cases, the 7-mer (X1 X2 X3 X4 X5 X6 X7) comprises TALKPFL. Insome instances, the 7-mer comprises TTLKPFL. In some instances, the7-mer comprises TLQIPFK. In some instances, the 7-mer comprises TMQKPFI.In some instances, the 7-mer comprises SIERPFK. In some instances, the7-mer comprises RYQGDSV.

In some instances, the AAV capsid protein comprises an insertion of atleast or about three, four, five, six, or seven amino acids of an aminoacid sequence T-X2 L K P F L at an amino acid position 588_589 in aparental AAV9 capsid protein (SEQ ID NO: 1), wherein X2 is A or T. Insome cases, the AAV capsid protein has an increased specificity forviral transduction in brain vascular cells (GLUT1+), as compared to areference AAV (e.g., AAV9). In some cases, the AAV capsid protein hasincreased specificity for viral tranduction in astrocytes, as comparedto a reference AAV (e.g., AAV9).

In some instances, the AAV capsid protein comprises an insertion of atleast or about three, four, five, six, or seven amino acids of an aminoacid sequence T-X2-Q-X4-P-F-X7 at an amino acid position 588_589 in aparental AAV9 capsid protein (SEQ ID NO: 1), wherein X2 is L or M; X4 isI, K, or L; X7 is K or I. In some cases, the AAV capsid protein hasincreased specificity for viral transduction in neurons and astrocytes,as compared to a reference AAV (e.g., AAV9). In some instances, theamino acid sequence is TLQIPFK. In some instances, the amino acidsequence is TMQKPFI. In some instances, the amino acid sequence isTLQLPFK.

In some instances, the AAV capsid protein comprises an insertion of atleast or about three, four, five, six, or seven amino acids of an aminoacid sequence S-I E R P F K at an amino acid position 588_589 in aparental AAV9 capsid protein (SEQ ID NO: 1). In some cases, the AAVcapsid protein has increased specificity for viral transduction inneurons and astrocytes, as compared to a reference AAV (e.g., AAV9).

In some instances, the AAV capsid protein comprises an insertion of atleast or about three, four, five, six, or seven amino acids of an aminoacid sequence R-Y-Q-G-D-S-V at an amino acid position 588_589 in aparental AAV9 capsid protein (SEQ ID NO: 1). In some cases, the AAVcapsid protein has increased specificity for viral transduction inastrocytes, as compared to a reference AAV (e.g., AAV9).

TABLE 2 List of 7-mer targeting peptides that can targetthe CNS with greater efficiency and specificity Brain- SEQ SEQ 7 mermean- ID Sequence (7 mer Amino ID Amino enrich NO acid) NO acid (log10)10 ACGTTGCAGATTCCTTTTAAG 435 TLQIPFK 2.730563 11 ACCGCCCTCAAACCCTTCCTC436 TALKPFL 2.536397 12 ACCACCCTCAAACCCTTCCTC 437 TTLKPFL 2.513839 13AGCATCGAAAGACCCTTCAAA 438 SIERPFK 2.357132 14 ACCCAAAACAGACCCTTCCTC 439TQNRPFL 2.22812 15 ACCATGCAAAAACCCTTCATC 440 TMQKPFI 2.165359 16ACTAGTACGCGGCCGTTTTTG 441 TSTRPFL 2.14357 17 GGCACCTTCGTCCCCCCCACC 442GTFVPPT 2.110151 18 TGGTCGACTAATGCGGGTTAT 443 WSTNAGY 1.979177 19ACCCTCGAAAGACCCTTCACC 444 TLERPFT 1.892846 20 ACTGCTGCTAAGCCGTTTCTG 445TAAKPFL 1.863541 21 ATTAGGATTGGTTATTCGCAG 446 IRIGYSQ 1.835644 22GAGCGTGTGGGTTTTGCTCAG 447 ERVGFAQ 1.73803 23 GCCGACCTCCTCAACTACAGA 448ADLLNYR 1.599571 24 CTCGTCGCCGGCTTCAGCCAA 449 LVAGFSQ 1.591793 25TCGTCTCTGAAGCCTTTTCTG 450 SSLKPFL 1.574365 26 ACGCATTCTAGGCCGTTTATT 451THSRPFI 1.566468 27 CCTTTTCCTGGTTATTCTGCG 452 PFPGYSA 1.555967 28ATTATTGTTGGGTATAGTCAG 453 IIVGYSQ 1.515858 29 AGGTATCAGGGTGATTCTGTT 454RYQGDSV 1.486197 30 AGGTATGCTGGGGATTCTATT 455 RYAGDSI 1.456352 31AAGACGATTGGGGAGCATTGG 456 KTIGEHW 1.44094 32 TGGATGACGCATGGTTCTGCG 457WMTHGSA 1.432726 33 GCTGCTTCGAGGCCGTTTCTT 458 AASRPFL 1.3791 34ACTACTTCTAGGCCGTTTCTT 459 TTSRPFL 1.353519 35 GCTCGTGTGGGTTTGGCTCAG 460ARVGLAQ 1.345499 36 GGGACTAGTCCGAATCTTGCG 461 GTSPNLA 1.306429 37GTGCGTACGGGGTATAGTTCT 462 VRTGYSS 1.302979 38 ATTCGGGTGGGGTATAGTTCT 463IRVGYSS 1.286795 39 ACTACTTTGCGTCCGTTTATT 464 TTLRPFI 1.284391 40GGTATTACGTCTCTGTTTAAG 465 GITSLFK 1.251837 41 ACCACCCAAACCCCCTTCAGA 466TTQTPFR 1.192121 42 ACTGTGTCGAGGCCGTTTTTG 467 TVSRPFL 1.171604 43TATGGGACGAATCCGTTTCGT 468 YGTNPFR 1.13664 44 CCCACCATGGTCGACAGCAGC 469PTMVDSS 1.099202 45 AATAAGGCTAGTTCTCAGAAT 470 NKASSQN 1.084711 46ACTTTTCGTAGTTCGAATGAT 471 TFRSSND 1.032118 47 CTGCCGGATCGTTATCCTAAT 472LPDRYPN 1.019382 48 CAGAATGTTACGAAGGGTGTT 473 QNVTKGV 1.000865 49GCTCAGTCTCCGATTCTTCGG 474 AQSPILR 0.979922 50 GTGGCTACTGGTTATTCTTCG 475VATGYSS 0.978116 51 TTGAATTCGAATCGGCCTAAT 476 LNSNRPN 0.9672 52ACGATTCGTAATAATACGATT 477 TIRNNTI 0.959641 53 GATCTTAGTCGTATTTTTAAG 478DLSRIFK 0.950386 54 CTCAGCAACAGCGCCAGCCAA 479 LSNSASQ 0.941212 55CTCGAAAACATGGTCATGAGC 480 LENMVMS 0.937552 56 TTGACTCAGCAGACGGATGTT 481LTQQTDV 0.936974 57 GCCGGCCTCGCCCACGCCACC 482 AGLAHAT 0.936046 58AATGCTCATACTGCGCTGGAT 483 NAHTALD 0.933184 59 TGGCCGATTCTTCGGGCTGAT 484WPILRAD 0.931545 60 GCGCTGGCTGGTCTGTCTCTG 485 ALAGLSL 0.930454 61CGTATTGATATGCCTTTTAAG 486 RIDMPFK 0.920205 62 ACGATGTCGTTGAGTAGTAGT 487TMSLSSS 0.919718 63 CAAAACGTCGCCAAAAACCTC 488 QNVAKNL 0.916602 64TTCGACAACCCCAACAACGTC 489 FDNPNNV 0.915503 65 GTCCTCATGAGAGAAAGCCTC 490VLMRESL 0.914386 66 CACCACGACACCGGCAGCGCC 491 HHDTGSA 0.912951 67GATGCGCCGAATAGTGTGGAT 492 DAPNSVD 0.901999 68 ACTGCGAGTAGGGATGCTAAG 493TASRDAK 0.889552 69 ACTAATTATACGAAGCAGATT 494 TNYTKQI 0.889024 70CCTCATAATGCTTCTGTGTTG 495 PHNASVL 0.864707 71 ACCAACAACACCAGAAACATC 496TNNTRNI 0.862873 72 ATGGTTCCTAATCATTCTGGT 497 MVPNHSG 0.859604 73CCGGTGCCGCAGTCTGAGCAT 498 PVPQSEH 0.852761 74 CTCTACCACGGCGGCAGCACC 499LYHGGST 0.851158 75 ATGATTCATACTAGGGAGACG 500 MIHTRET 0.850116 76ACTAATAATACGAAGCCTCTT 501 TNNTKPL 0.849557 77 CTCAACACCACCAGAAACACC 502LNTTRNT 0.839304 78 GCTTTGCATAATTCTCAGAAT 503 ALHNSQN 0.827469 79GTGAATAGTACGAGGAATGTT 504 VNSTRNV 0.826565 80 GGTGTTGGGGTTGTTACGAAG 505GVGVVTK 0.820556 81 GTTTTGTCGAAGCAGTTGGCT 506 VLSKQLA 0.816495 82TCTAAGTCTCTGCAGCCGTTG 507 SKSLQPL 0.815502 83 TCGAATTCTACGCGGTTGGTT 508SNSTRLV 0.815119 84 CTCAACCAAACCAAACAAATC 509 LNQTKQI 0.811665 85AACAACAGCGTCAGACAACTC 510 NNSVRQL 0.806765 86 ATGTCGGGGTATTCGCATACT 511MSGYSHT 0.804194 87 GTCAAAGTCCCCATCATCACC 512 VKVPIIT 0.799412 88GGGAATAATACTCGGGCGGTG 513 GNNTRAV 0.797634 89 CAGAATCAGATTAAGAATATT 514QNQIKNI 0.797303 90 ACCAACATGACCAAACCCCTC 515 TNMTKPL 0.791064 91AACAACAGCACCAGACTCAGC 516 NNSTRLS 0.790429 92 AGCATGCTCACCAGCATCGTC 517SMLTSIV 0.790252 93 CTCAACGCCACCGCCAGCAGA 518 LNATASR 0.785269 94AATACTGGGGTGCAGGTTAGG 519 NTGVQVR 0.785026 95 ACCAACACCGTCAGACCCATC 520TNTVRPI 0.780569 96 CAGTCTACGCCGGGGGCTACT 521 QSTPGAT 0.779842 97GGTCATTCTGAGAATTCTCGT 522 GHSENSR 0.778724 98 CTCCCCACCCAAACCCTCAGA 523LPTQTLR 0.773756 99 AAGAATGCTCCGATGCCTGAT 524 KNAPMPD 0.772397 100AGCAACAGCACCAAACCCACC 525 SNSTKPT 0.768053 101 CCTATTATTACTATGCCTGCT 526PIITMPA 0.767318 102 CCCCTCAAAGGCATCGGCAGC 527 PLKGIGS 0.767253 103GTCCTCAGCGTCAACCACCTC 528 VLSVNHL 0.765014 104 AGTGTGGCTGCTGTGCATATG 529SVAAVHM 0.764013 105 AGCAACAGCATCAGACTCGTC 530 SNSIRLV 0.763114 106CATAATGATACTCGTCCTCTT 531 HNDTRPL 0.759962 107 CCTCCGGTGAGGTCTACGGCT 532PPVRSTA 0.755777 108 ATGAACACCGTCAGAAACCTC 533 MNTVRNL 0.754463 109TTGACGGATGGTCATCGGATT 534 LTDGHRI 0.754429 110 ACGAATGCGGTGAAGCTGACT 535TNAVKLT 0.752766 111 CGTGTGGATGCGGTTTTGAGT 536 RVDAVLS 0.750458 112ACCAACGCCGTCAAAAGCACC 537 TNAVKST 0.749271 113 ATGCTCAACACCACCGCCAGA 538MLNTTAR 0.747921 114 GGCAACAACACCAGACCCACC 539 GNNTRPT 0.747523 115CAGTCTGGGGGTAGTGCGTTG 540 QSGGSAL 0.744332 116 GTGATTGCTTTGTCTACTATG 541VIALSTM 0.743901 117 CTGAATACGATTCGGAATGTG 542 LNTIRNV 0.743811 118AGCAACGCCACCAGAAGCGTC 543 SNATRSV 0.740235 119 GGCAACGGCACCAAATTCATC 544GNGTKFI 0.736944 120 AGCAACACCATCAGACCCGTC 545 SNTIRPV 0.736197 121AGCAACAGCACCAGAGCCATC 546 SNSTRAI 0.734716 122 TTTCATTTGACTGATAGGGGT 547FHLTDRG 0.733831 123 CCGATTAGGTCGGATACTGTG 548 PIRSDTV 0.731286 124GCCAACCAAATCCACAACACC 549 ANQIHNT 0.730071 125 TCGAATACGGTTCGGAATACT 550SNTVRNT 0.727807 126 ACCAGCCTCGGCTACAGCAGC 551 TSLGYSS 0.727167 127ATCAGCGGCGTCCAAACCAGA 552 ISGVQTR 0.724309 128 TCGAATGCTGTTAGGCAGACT 553SNAVRQT 0.72361 129 TCTCTTTCTGAGCTTAGGATT 554 SLSELRI 0.723061 130AGTACTGAGAAGAGGGATGAG 555 STEKRDE 0.721349 131 AACCTCGTCGGCACCCTCATC 556NLVGTLI 0.72103 132 GGCAACAACATCAGACTCACC 557 GNNIRLT 0.719643 133GCCGTCCCCACCGGCCTCAGA 558 AVPTGLR 0.717951 134 GGTGATAATAGTCTGACTAGG 559GDNSLTR 0.717624 135 CACGCCGACAACAGACTCAAA 560 HADNRLK 0.714815 136AGCAACGCCACCAGAAACTTC 561 SNATRNF 0.714052 137 GGGAATACGACTAGGGGGCTG 562GNTTRGL 0.713945 138 CTCAACCTCACCGCCACCAAC 563 LNLTATN 0.71358 139AGCGGCGTCAGCACCACCGAC 564 SGVSTTD 0.711249 140 TATGCTAAGACGTTGGCTATG 565YAKTLAM 0.710703 141 ATGAACCACACCAAACCCACC 566 MNHTKPT 0.710702 142GCTAATGCTACGAGGAATTCT 567 ANATRNS 0.707423 143 CAAAACCAAACCAAAATCACC 568QNQTKIT 0.707235 144 TCTAATGCTATTAAGGCTACG 569 SNAIKAT 0.70699 145TATCCTGATAATTATGGTAAG 570 YPDNYGK 0.706343 146 GGCAACACCACCATCAGAAGC 571GNTTIRS 0.705947 147 CTGGGTCTTACGGGTGCGGTT 572 LGLTGAV 0.705628 148GCGAATAGTACGCGTATTCTG 573 ANSTRIL 0.704068 149 GATAATGCGATTCGTGCGCAG 574DNAIRAQ 0.703945 150 ACGAATTATACTAAGCTTATG 575 TNYTKLM 0.702916 151CATAATAGTCCTAGTAGTTAT 576 HNSPSSY 0.70154 152 GAGAATATGGTTCGGTCTGTG 577ENMVRSV 0.700139 153 ACCAACAACATCAAAAGCTAC 578 TNNIKSY 0.698201 154ACTAATTCGACGAGGCCTGTG 579 TNSTRPV 0.696679 155 CATACGGCTCCGCCTCATCCT 580HTAPPHP 0.695156 156 GCCCTCAGCCAAAACACCAGC 581 ALSQNTS 0.692869 157CCGAATGTGACTAAGAATGCT 582 PNVTKNA 0.692038 158 CTTGATACGCTGACGGGTTAT 583LDTLTGY 0.690142 159 CCTAATAGTGTGCGTTCTGTT 584 PNSVRSV 0.689082 160ACCAGCAGCCACCTCCCCCAC 585 TSSHLPH 0.688469 161 GTTCTTCATGGTGGTAATGAT 586VLHGGND 0.687991 162 AGCAACTACGTCAAAAGCGCC 587 SNYVKSA 0.687276 163GACAACAACAGCACCAGATGG 588 DNNSTRW 0.687057 164 ACTGTGATGAAGGGTTTTACT 589TVMKGFT 0.687036 165 AGCCACACCCTCAGCACCCTC 590 SHTLSTL 0.686592 166CCTGGTCCTCAGCAGGCGAAG 591 PGPQQAK 0.685365 167 ACTTTTGGGACGTCTAAGTTG 592TFGTSKL 0.684153 168 AGCAACGCCGTCACCAACAGA 593 SNAVTNR 0.683838 169TTGTCTATGTCGACGGTGCCT 594 LSMSTVP 0.682136 170 AGGCGTTATGATGGTAGGGAG 595RRYDGRE 0.681722 171 ACCAACAGCACCAAAAACATC 596 TNSTKNI 0.678439 172ATTAATGCTCAGTGGTCTGCG 597 INAQWSA 0.672085 173 GCCCAAACCAGCAACGACCCC 598AQTSNDP 0.669325 174 ACGAATGAGACTAGGATGGTG 599 TNETRMV 0.669307 175GCCAACGCCACCAGAGGCGTC 600 ANATRGV 0.667153 176 GCGGATGTGAATGCTTCGGGT 601ADVNASG 0.666574 177 CTTAGTGAGCGGAATTATGTG 602 LSERNYV 0.665675 178AGTGGTGAGCTTGCGCGGGCG 603 SGELARA 0.663381 179 GGGAATACTGCTAAGAATATT 604GNTAKNI 0.662988 180 GACAACGCCGTCAGACCCCTC 605 DNAVRPL 0.662435 181ATGATGCTCAGCGGCCTCAAC 606 MMLSGLN 0.660837 182 CCCAACACCATCAGAAACGTC 607PNTIRNV 0.660528 183 GAGAATTTGACGCGTGGGGTG 608 ENLTRGV 0.659884 184TTTAGTGCTCGGAGTACGGGG 609 FSARSTG 0.659095 185 GAAAACGCCACCAGAACCTAC 610ENATRTY 0.657305 186 GGCAACAGCACCAGAATGAAC 611 GNSTRMN 0.656999 187GGGAATTCTACTAAGTCTCCT 612 GNSTKSP 0.656908 188 ATGCAAACCACCCTCCACCTC 613MQTTLHL 0.656525 189 AACCCCACCCTCATCACCCTC 614 NPTLITL 0.655924 190CTTATTTCTGGTCATGCGCAG 615 LISGHAQ 0.655121 191 TTCAACCAAGTCAGAAGCCTC 616FNQVRSL 0.65493 192 ATGAACATCCTCAGAGAAGTC 617 MNILREV 0.654097 193AGTGCTATGATGCGTGGTGTT 618 SAMMRGV 0.653566 194 CCCATGCTCAGCAGCCCCAGC 619PMLSSPS 0.652269 195 CAGAATAATACGCTTAGGTCG 620 QNNTLRS 0.651653 196AGGCATTCGGATCCGGTGGAG 621 RHSDPVE 0.65144 197 GTGAATACGACGTTTTCTACG 622VNTTFST 0.65142 198 ATCAACAGCACCAGAGGCATC 623 INSTRGI 0.650374 199ACCAACGCCGTCAGAGACCTC 624 TNAVRDL 0.649865 200 TCTAATAGTACTAGGCTGTCG 625SNSTRLS 0.649567 201 ACGTCTATTGCTGGGTCGTTT 626 TSIAGSF 0.647793 202CTCGGCAGCACCTACCCCAGA 627 LGSTYPR 0.644533 203 GCCAACGTCACCACCCAAAGA 628ANVTTQR 0.643299 204 AGCAACAGCGTCAGACTCAGC 629 SNSVRLS 0.641635 205GCTACGAATGAGCCTGATCGG 630 ATNEPDR 0.641008 206 AGAATGAACCCCGAACAAAGC 631RMNPEQS 0.640866 207 TCGGTTAGTGGTTCGGCTAAT 632 SVSGSAN 0.640362 208CCGAGTAATAGTACTACGCGT 633 PSNSTTR 0.639664 209 CTTTTTCATGGTACGCATGAG 634LFHGTHE 0.639176 210 CCCCAACTCGGCAGCACCAAA 635 PQLGSTK 0.638022 211CCCATCAGCGCCAACAGACTC 636 PISANRL 0.637814 212 AATTCGGGTAATCTTTCTATG 637NSGNLSM 0.637206 213 AAGGTTGAGGCTATTGGGATG 638 KVEAIGM 0.63585 214AAGACTTTGCTTAATAGTGTT 639 KTLLNSV 0.635567 215 AATGCTATGGATCTTAAGGTG 640NAMDLKV 0.633592 216 ATGATGGGTAATGGGTCTTCG 641 MMGNGSS 0.632846 217AGCAACAGCACCAAAGCCCTC 642 SNSTKAL 0.630646 218 TCTAATAGTACGCGGGGTACG 643SNSTRGT 0.630359 219 AGCCTCAGCAGCCTCCACGTC 644 SLSSLHV 0.629137 220ACCCTCAGCGGCGCCCTCACC 645 TLSGALT 0.628275 221 CCGAATACGGTGAGGAATAAT 646PNTVRNN 0.627943 222 AATAATAATGTGAAGATGACT 647 NNNVKMT 0.627302 223TCTGTTGCTAAGCCTTTTATG 648 SVAKPFM 0.627075 224 ACGCAGACGACTCGGCTTTCG 649TQTTRLS 0.625657 225 AATAATGCGACTCGGGGTGGG 650 NNATRGG 0.622765 226ACCAACACCACCGCCAGAATC 651 TNTTARI 0.621665 227 GAGAATAGTATTCGGACTATT 652ENSIRTI 0.619021 228 ATTAGGGAGACTTCTGGGAAG 653 IRETSGK 0.618732 229AGCATCTACTACCACACCGAA 654 SIYYHTE 0.618441 230 GTCCTCGACAACAGCGGCCAA 655VLDNSGQ 0.618183 231 AGCCACAACATGACCATCAGA 656 SHNMTIR 0.614114 232ACCATCCCCAGCGCCGGCAAA 657 TIPSAGK 0.612816 233 CCTAGGCATACTTTGAGTCAG 658PRHTLSQ 0.60799 234 TCGCTTAATGTGCAGGATGTG 659 SLNVQDV 0.607203 235TATCAGTCTCTGGCTAATCCG 660 YQSLANP 0.607005 236 ATTCTGAATATGTCTACGGAT 661ILNMSTD 0.606834 237 GTCCTCCACCTCGGCCACAGC 662 VLHLGHS 0.605932 238GGTGTTACTCAGACTCCGCGT 663 GVTQTPR 0.6059 239 ACCAACGACACCAGAAGAATC 664TNDTRRI 0.604587 240 CAAACCGACCACACCAGCAGA 665 QTDHTSR 0.604031 241AAGAATGAGATTAAGAATGTG 666 KNEIKNV 0.603838 242 TGGACTGGTAATGAGAGGCTT 667WTGNERL 0.603412 243 GGTAAGAGTGATGCTATGCGG 668 GKSDAMR 0.603322 244GGGAATGCGACTCGTACTTAT 669 GNATRTY 0.602016 245 GCCAGCAACCTCGGCCTCCCC 670ASNLGLP 0.600647 246 AGCAGCCTCAGCGGCAGCCCC 671 SSLSGSP 0.599424 247GCCGCCGTCAACCAAGGCGTC 672 AAVNQGV 0.599283 248 GACCAAAACCAACCCAGAGAA 673DQNQPRE 0.59872 249 CTCACCCAAGACAAACAAGCC 674 LTQDKQA 0.59843 250TTCGGCAGCAACGAACACAAC 675 FGSNEHN 0.59773 251 CTGAATCAGCCGAATGTGCGG 676LNQPNVR 0.59703 252 ATGAATACTACGCGTAATCTG 677 MNTTRNL 0.596819 253ACTAATTCTACTAGGACGAGT 678 TNSTRTS 0.596817 254 TTTGAGCTTTCGCATGTGCCT 679FELSHVP 0.595373 255 GAAGGCCTCGGCAACGCCGCC 680 EGLGNAA 0.59339 256CTCACCGTCACCCTCAACCAC 681 LTVTLNH 0.593104 257 TTTGGGAATGCGATTCAGTCT 682FGNAIQS 0.593063 258 CCGGGTGGGGGTTTGACTCCG 683 PGGGLTP 0.591748 259CCCAACATGACCAGAAGCCTC 684 PNMTRSL 0.591633 260 AGTCAGCTGCATAGGTTGCAG 685SQLHRLQ 0.588997 261 AATGATCCTGTGCTGACTGTG 686 NDPVLTV 0.587622 262GAAAACAGCGTCAGAACCACC 687 ENSVRTT 0.586872 263 GATCGTGTGACTAATCCGAAG 688DRVTNPK 0.585898 264 CTCAACACCGGCCAAATGAGA 689 LNTGQMR 0.584482 265ATTAGTCCTTCTCAGGTGAAG 690 ISPSQVK 0.583565 266 TACAGCGCCGACAAAACCACC 691YSADKTT 0.583168 267 ATTGATAGTGCGGGGATGGCG 692 IDSAGMA 0.582726 268AGCAACAACATCAAACTCAAC 693 SNNIKLN 0.582501 269 CTTGAGACGCAGCCTAGGACT 694LETQPRT 0.582054 270 AATTTGCATAGTAATGTGCTG 695 NLHSNVL 0.581653 271ATGGTCAAAGACACCCAACTC 696 MVKDTQL 0.581409 272 CTCATGCACCTCACCAACCCC 697LMHLTNP 0.579725 273 GTCGCCAGCCCCGACAAAAGA 698 VASPDKR 0.579259 274GTCGTCGCCGTCCTCACCAGC 699 VVAVLTS 0.578391 275 AACATCATGGTCAACGTCCCC 700NIMVNVP 0.578008 276 CCGTATAGTGGGCAGCGGACT 701 PYSGQRT 0.577882 277CCTCTGCTTAAGACGAATACT 702 PLLKTNT 0.575523 278 GGGAATACTACGGTGCGTGGG 703GNTTVRG 0.574402 279 ACCAACAAAGTCAGAGACGAC 704 TNKVRDD 0.573889 280CCCAGCACCGCCCTCCTCGTC 705 PSTALLV 0.573034 281 ATCCAAAGCATCACCCTCAAA 706IQSITLK 0.572783 282 CCCCTCCACGCCAACATGAGC 707 PLHANMS 0.570881 283ACCGCCCACGCCGAAGCCAGA 708 TAHAEAR 0.570572 284 ACGAATACGGATTCGTATCGT 709TNTDSYR 0.569365 285 ACCCACGTCAGCCTCGACAGA 710 THVSLDR 0.569228 286TTTAGTACGAAGGATCATGTT 711 FSTKDHV 0.568654 287 GACAACACCCAAACCGCCCCC 712DNTQTAP 0.568553 288 AGCAACAGAATCATCAGCGGC 713 SNRIISG 0.568551 289ACTAATTCTGTTAGGAATAAT 714 TNSVRNN 0.568237 290 AGCAACGAAGTCAGAAACATG 715SNEVRNM 0.567847 291 GAACTCTACAAACCCAGCAGA 716 ELYKPSR 0.567258 292TGGATTACGGGGGGTGCGAGT 717 WITGGAS 0.567159 293 AACAACAGCGTCAGACCCACC 718NNSVRPT 0.566945 294 CACACCGCCGTCCTCAGAACC 719 HTAVLRT 0.565911 295AATACTTTGTCTCTTGCTCCT 720 NTLSLAP 0.564955 296 CCCAACGCCAGCGTCAACAGC 721PNASVNS 0.564885 297 GTTACTGGTGGGAATACTTAT 722 VTGGNTY 0.564099 298GTCAACGAAAACCTCATCGAA 723 VNENLIE 0.563902 299 GTTTTGACGACTCATTCGAAT 724VLTTHSN 0.563552 300 CACACCCAACTCCCCCTCACC 725 HTQLPLT 0.562802 301CGGCAGTCTCTTGAGGCGTTG 726 RQSLEAL 0.562715 302 CCCGACGTCACCGAAGGCAGA 727PDVTEGR 0.560929 303 CCGACTAATATTATGCTTGAT 728 PTNIMLD 0.560313 304CCCAACATGAGCGCCATGATC 729 PNMSAMI 0.559451 305 ATTATGAATGGTCAGGCTCTG 730IMNGQAL 0.558677 306 AATAATAATTCGACGCGTTTT 731 NNNSTRF 0.558656 307GCCAACGTCACCAGAAGCACC 732 ANVTRST 0.558653 308 CAGAATCTTGGTCTTAATGTG 733QNLGLNV 0.558202 309 CAAGCCCAAATGAGCAGCGCC 734 QAQMSSA 0.557238 310CTCACCATGGCCACCAACGTC 735 LTMATNV 0.556779 311 GATCGTGATTCTGTGCAGAAT 736DRDSVQN 0.55654 312 GTCAACCCCACCAACAACCTC 737 VNPTNNL 0.555076 313CCGTCTTTTACGACTATGAGT 738 PSFTTMS 0.554811 314 AGGGAGACGCCTATTCCTAAG 739RETPIPK 0.553803 315 AGCCTCAACTTCACCACCGCC 740 SLNFTTA 0.553055 316GGTATGAGTGGGGTTGCTCAG 741 GMSGVAQ 0.552736 317 AAGGTTGATGCGGCTCAGAGT 742KVDAAQS 0.552569 318 CATATTACGACTAATGTGTCT 743 HITTNVS 0.552479 319GAGCGGGAGTCGGCTCGTCTT 744 ERESARL 0.551876 320 GATAATAGGACTCAGAGGACG 745DNRTQRT 0.551636 321 TCTGCGCCTAATGTGACTATT 746 SAPNVTI 0.551278 322GAAAACGGCACCAGAAACACC 747 ENGTRNT 0.55012 323 GGCACCGCCGTCTTCACCGCC 748GTAVFTA 0.549912 324 GCCAACAACATGAGCCAAACC 749 ANNMSQT 0.549519 325GGTAATCATACTTATAATCTG 750 GNHTYNL 0.549264 326 CTGAGTCCTTCGAATTCTAAT 751LSPSNSN 0.549019 327 ATGGGCGCCGACCACAGAACC 752 MGADHRT 0.548749 328AGTAATTCGACGCGTGGTTCT 753 SNSTRGS 0.548705 329 AATACTTCTGGTGTGCCTAAG 754NTSGVPK 0.548579 330 ATCAACACCGCCGTCGTCAGC 755 INTAVVS 0.54846 331ATGGGGGTTCAGGCTTATGTG 756 MGVQAYV 0.54845 332 GGGAATTTTGTTAAGCCGAAT 757GNFVKPN 0.547942 333 GTGAATTCGGTGAGGATGATT 758 VNSVRMI 0.547171 334CACGACAGCGCCAACAGCAGA 759 HDSANSR 0.54671 335 CTCGGCGTCAGAGACGACAGC 760LGVRDDS 0.545708 336 AGCAACGCCGTCAGAGCCAAC 761 SNAVRAN 0.545674 337CCCGTCCTCGCCGCCACCATG 762 PVLAATM 0.545458 338 AATAAGCCTGTGTCTGGTAAT 763NKPVSGN 0.544076 339 GTCGGCCTCGCCAGCCACACC 764 VGLASHT 0.541637 340GGCAGCAACACCAACATGAAA 765 GSNTNMK 0.540634 341 GTTGCTACTACTGTGCATAAT 766VATTVHN 0.540191 342 AACACCCTCGACAGCAGAGTC 767 NTLDSRV 0.539923 343AGGGAGGCTAATTTGCAGGCG 768 REANLQA 0.539717 344 GTCGACGCCCTCAGCCACATG 769VDALSHM 0.539694 345 CTTCGTCTTGCTGGTCTTGCT 770 LRLAGLA 0.539525 346ATTACGGTGACTAATTATTCG 771 ITVTNYS 0.539447 347 TGGGACAGCGGCAGCGGCGAA 772WDSGSGE 0.539198 348 TTCGCCAGCGAAACCGTCGCC 773 FASETVA 0.537906 349CAGAATGTTACGGGGACGAGG 774 QNVTGTR 0.537867 350 CTTAATGGGTTGAATGTGTCT 775LNGLNVS 0.537505 351 AATATGCTTAAGCAGAGTGAG 776 NMLKQSE 0.537354 352TTTAATGAGGTTCCGAAGGCG 777 FNEVPKA 0.537144 353 CTCGGCGACATCACCGGCTTC 778LGDITGF 0.537047 354 ATTTCGGCTTCTCATTCTCGT 779 ISASHSR 0.536983 355AGTCAGACGCAGATTGCTCTT 780 SQTQIAL 0.53684 356 AATATGGCTACTCAGATGAAG 781NMATQMK 0.536747 357 GTCAACCACAACATCAGACTC 782 VNHNIRL 0.53652 358AACGCCCTCCAAGTCCCCGTC 783 NALQVPV 0.536474 359 AATTCGACGGGTATTGATACG 784NSTGIDT 0.53526 360 GTTGTTGCTGGGCATCTTAAT 785 VVAGHLN 0.534827 361CGGAATGATGCGATTCTGAAT 786 RNDAILN 0.53456 362 AATCGGGTTGATTCGCGGGCT 787NRVDSRA 0.53408 363 TCGACGCATTCTACTTATGTG 788 STHSTYV 0.533753 364AGTGGGCATGGGACTCTGCGG 789 SGHGTLR 0.532267 365 CCCAACAGCACCACCCTCAGC 790PNSTTLS 0.531723 366 GACACCAAACCCACCAACACC 791 DTKPTNT 0.531589 367AACAGAGACACCATCAACAAC 792 NRDTINN 0.531212 368 CCCAGAACCCTCAGCGACGGC 793PRTLSDG 0.530962 369 ACCAAAGACGTCACCACCAGC 794 TKDVTTS 0.53023 370CTGGGTAATCCTACGCCTTCT 795 LGNPTPS 0.529624 371 TATCAGGCGATGTATAGGGAT 796YQAMYRD 0.528592 372 AGCGGCGTCCAAGAATTCGAC 797 SGVQEFD 0.528493 373TCTAATCATCTGTCGACGGTT 798 SNHLSTV 0.528046 374 CACGACGAAAGAGCCAACATG 799HDERANM 0.526932 375 ACGAATAGTACTCGGTCGCCT 800 TNSTRSP 0.526707 376CCGACGGATAAGTCTTATCCG 801 PTDKSYP 0.525687 377 ATGACCGAACAAAGAACCGCC 802MTEQRTA 0.524729 378 ACCAACGCCACCCACAGCAAA 803 TNATHSK 0.523113 379AATCGGGGTACTTTTAGTGCG 804 NRGTFSA 0.522986 380 AACACCACCAAATTCAACACC 805NTTKFNT 0.522899 381 GGGATTCCGCCTTTGACTAAG 806 GIPPLTK 0.522659 382ACCGCCAACAGCACCCAAAGA 807 TANSTQR 0.522626 383 ACTCAGGGTCCGGGTCCGAAG 808TQGPGPK 0.522239 384 ATGAATACTACGCGGAATTAT 809 MNTTRNY 0.522193 385CTCAGAGAAGGCACCTTCATG 810 LREGTFM 0.521554 386 AACATCAGCAGCACCGACAGA 811NISSTDR 0.521154 387 TCGGTTAGTCGGCCGTTTCAG 812 SVSRPFQ 0.520365 388CCTATGTCTGCTAATGGGAAG 813 PMSANGK 0.518792 389 GAAGTCAGAGGCAACACCTTC 814EVRGNTF 0.518514 390 TCGCAGAAGGGTCTGTCTGTG 815 SQKGLSV 0.518477 391ATTGCTGAGAATCTGAGTGCT 816 IAENLSA 0.517935 392 TTCAGCAACATGAACCTCAAA 817FSNMNLK 0.517734 393 CCTCGTGAGCGGACTTATACT 818 PRERTYT 0.516835 394TGGTCGCATAATCCGGATTCT 819 WSHNPDS 0.516248 395 GGTCTTCAGACTCTGATTACG 820GLQTLIT 0.516123 396 AGCAACAGAACCAAAGACATC 821 SNRTKDI 0.515515 397GTGACTGAGACGATTCTTAAG 822 VTETILK 0.515459 398 TATGTTACGCGTAGTGGTCTG 823YVTRSGL 0.515319 399 ATGAACGCCAACATCCTCGTC 824 MNANILV 0.514679 400ACTACGAATCGGGTGGGTACG 825 TTNRVGT 0.51427 401 GTCCTCAAAAGCCACCTCCAA 826VLKSHLQ 0.51406 402 AGCGACGGCCTCAGAACCCAA 827 SDGLRTQ 0.514048 403CTCAACAGCGTCAAACCCAAC 828 LNSVKPN 0.513525 404 AGCCAAGCCATCGCCCCCAAA 829SQAIAPK 0.51343 405 TCTAATAATGTTCGTGCTCCG 830 SNNVRAP 0.513257 406ATCAGCCACCTCAGCGAAAGC 831 ISHLSES 0.511503 407 CTCCTCCCCGGCATGAGATTC 832LLPGMRF 0.5114 408 CTTCTTGCGTCGGCTACGAAG 833 LLASATK 0.510718 409AACCACAACGGCATGGTCAGC 834 NHNGMVS 0.510709 410 CACAGCGGCGAACTCAACAAC 835HSGELNN 0.510488 411 CTTTCTGCGCCTAAGGATACT 836 LSAPKDT 0.509967 412ATTGGGACTCATGTGGGGACG 837 IGTHVGT 0.509748 413 GCCGTCGCCCTCGCCAGCGCC 838AVALASA 0.509614 414 ATTACTCTTCAGTCTAATGCT 839 ITLQSNA 0.509556 415GTCGACCACAGCCTCACCAGC 840 VDHSLTS 0.509193 416 GGCAACGAAGCCACCAGCCTC 841GNEATSL 0.508729 417 CCCGTCAGCAGCACCACCCTC 842 PVSSTTL 0.507682 418CCCTACCAAACCAGCAGCGCC 843 PYQTSSA 0.507351 419 ACCAGCGTCGTCAGCGCCTTC 844TSVVSAF 0.507058 420 AACGGCGTCCCCGACAACAAC 845 NGVPDNN 0.50693 421AGCACCCAAACCATCGGCTTC 846 STQTIGF 0.505905 422 ATTCGGAGTTCTGATCTTGCG 847IRSSDLA 0.505273 423 AACACCCAAAGCGCCAAATAC 848 NTQSAKY 0.504002 424AGTCTTGAGTCGTCTAGGCAG 849 SLESSRQ 0.503575 425 CTCAACCTCGCCAGCGGCAGA 850LNLASGR 0.503463 426 ATGCAGGAGCGTCGGGAGTAT 851 MQERREY 0.503055 427TGGCTCCTCAAAGACGGCTAC 852 WLLKDGY 0.502893 428 GGCAGATACCAAACCACCAGC 853GRYQTTS 0.502776 429 AATACGACGAAGTTTCCGTTT 854 NTTKFPF 0.502408 430ATGAGCAACGGCATCAGCAGA 855 MSNGISR 0.501897 431 CCGATTCGGGATCCGGAGAAG 856PIRDPEK 0.501479 432 AGTACGCTGAGTCCGCATGTT 857 STLSPHV 0.501315 433TTCATGCCCATCAGCAGCGCC 858 FMPISSA 0.50124 434 AGAAGCGACACCCAAAGCAGC 859RSDTQSS 0.500462

TABLE 3 11-mer targeting peptides that can target the CNS Mean SEQ SEQenrichment ID ID 11-mer Amino score (log 10 NO 11-mer DNA Sequence NOAcid Sequence scale) 860 GCCCAAACCACCCTCAAACCCTTCAGCAACC 864 AQTTLKPFSN1.101507461 CC P 861 GACGGCACCACCCTCAAACCCTTCAGCAACC 865 DGTTLKPFSN0.172425316 CC P 862 GACGGCACCGCCCTCAAACCCTTCCTCGCCC 866 DGTALKPFLA0.647416071 AA Q 863 GATGGGACGACTCTTAAGCCGTTTCTGGCAC 867 DGTTLKPFLAsequence AG Q predicted based on PHP.eB sequence

AAV Capsid Proteins Targeting the Liver

Disclosed herein are AAV capsid proteins with a substitution or aninsertion of at least one amino acid at an amino acid position describedabove in a parental AAV capsid protein that confers an increasedspecificity for the liver. In some instances, the insertion comprises atleast one amino acid provided in any one of the sequences provided inTable 4 and/or FIG. 35. In some instances, the insertion comprises atleast two amino acids provided in any one of the sequences provided inTable 4 and/or FIG. 35. In some instances, the insertion comprises atleast three amino acids provided in any one of the sequences provided inTable 4 and/or FIG. 35. In some instances, the insertion comprises atleast four amino acids provided in any one of the sequences provided inTable 4 and/or FIG. 35. In some instances, the insertion comprises atleast five amino acids provided in Table 4 and/or FIG. 35. In someinstances, the insertion comprises at least six amino acids provided inTable 4 and/or FIG. 35. In some instances, the insertion comprises atleast seven amino acids provided in Table 4 and/or FIG. 35. In someinstances, the amino acids are contiguous. In some instances, the aminoacids are not contiguous. In some instances, the insertion is at anamino acid position 588_589 in a parental AAV capsid protein. In someinstances, the parental capsid protein is AAV9 capsid protein, providedin SEQ ID NO: 1.

TABLE 4 List of 7-mer targeting peptides that target the liver SEQ IDSEQ ID 7 mer amino liver-mean- NO DNA sequence for 7 mer amino acid NOacid enrich (log10) 868 GCCGAATACAACACCGGCGTC 950 AEYNTGV 0.585999 869GCTAATGCGATGGGTGATCAT 951 ANAMGDH 0.611245 870 GAAAGCATCCAACAACTCAGC 952ESIQQLS 0.525712 871 TTTAAGGTTAGTATTCAGCAG 953 FKVSIQQ 0.591275 872TTTCTTCTTACTTCGGATCCG 954 FLLTSDP 0.515975 873 TTTCAGCAGGATACTTATCTG 955FQQDTYL 0.548808 874 TTTTCTGGTAGTGCTAGGGTT 956 FSGSARV 0.509852 875TTCAGCGTCACCAGACAAGCC 957 FSVTRQA 0.718621 876 GGGGCTGCTGGTAAGAGTTTG 958GAAGKSL 0.571307 877 GGTCATATTTCGGCGCTTGCG 959 GHISALA 0.552655 878GGGCTGAAGGTGTCTGGGTCT 960 GLKVSGS 0.777335 879 GGTCTTCAGTCGCCGAGGTCT 961GLQSPRS 0.572095 880 GGGAGGACGGTGCAGGATAGG 962 GRTVQDR 0.528631 881GGCACCAAAACCCACAGCCTC 963 GTKTHSL 0.518459 882 GGCACCACCAGAAGCCCCACC 964GTTRSPT 0.560705 883 CACCCCAGCCTCAGACAAAGC 965 HPSLRQS 0.508478 884CATTCTGCGAAGACTATTGCG 966 HSAKTIA 0.579949 885 CACAGCGGCCCCACCAGCAGC 967HSGPTSS 0.505295 886 ATCAACAAAGACAACAACCAA 968 INKDNNQ 0.531458 887ATCAACACCGCCAGCTACAAA 969 INTASYK 0.542125 888 AAAGCCATCAGCACCGCCAAC 970KAISTAN 0.616958 889 AAAGCCAACAGCCAAATCACC 971 KANSQIT 0.519425 890AAAGCCTACAGCGTCCAAGTC 972 KAYSVQV 1.15776 891 AAACTCAGCACCCTCCACCAA 973KLSTLHQ 0.568791 892 AAGCGGGCGCCTGCTGATTCT 974 KRAPADS 0.611303 893AAAACCAACAACACCGTCCTC 975 KTNNTVL 0.504377 894 AAAACCCAACTCGGCCAAATG 976KTQLGQM 0.519843 895 CTTGGTTCTGCGCTTACGAGG 977 LGSALTR 0.563314 896CTCATCAGCACCACCCACAGA 978 LISTTHR 0.502083 897 CTTAAGGTTGCGTCTGGTTTG 979LKVASGL 0.540598 898 CTTCTTGCGACTGGGCTTAAG 980 LLATGLK 0.545453 899CTTCCGGGTGGTGCGAGGTTG 981 LPGGARL 0.564086 900 CTTAGGGGTTCTGCTCAGGTG 982LRGSAQV 0.648123 901 TTGTCTGCTCAGCTTCCGCGG 983 LSAQLPR 0.503052 902CTTTCTACTGCGTTGACTGTG 984 LSTALTV 0.591029 903 CTCACCAGAGTCGGCACCGTC 985LTRVGTV 0.520831 904 ATGGCCAACACCAGAAGCGCC 986 MANTRSA 0.525721 905ATGATCAGAGGCACCAGCAGC 987 MIRGTSS 0.512891 906 ATGCTCAGCGGCCAAGCCAGA 988MLSGQAR 0.668364 907 ATGAGAGGCCAAGGCGTCCAC 989 MRGQGVH 0.567657 908AACAACAACCAAAAAGTCTAC 990 NNNQKVY 0.610002 909 AACAACAGCGGCCACATCAGC 991NNSGHIS 0.518228 910 AACACCCTCATCAACGTCAGC 992 NTLINVS 0.607212 911AATACTTCTGGTGTGCCTAAG 993 NTSGVPK 0.684638 912 AATGTGCCGACTTCGCCGCGG 994NVPTSPR 0.520245 913 CCCGCCAGAATCCCCGGCAGC 995 PARIPGS 0.50309 914CCCCTCAGAAACATCAGCGCC 996 PLRNISA 0.50497 915 CCGTTGAGTGGGGGTGTTCGT 997PLSGGVR 0.598554 916 CCCAACAGAGCCGCCAACAAC 998 PNRAANN 0.508143 917CCGCGTCATGCGACTCAGTCG 999 PRHATQS 0.711312 918 CCGCGGCCGGTGTCGAATGGG1000 PRPVSNG 0.7305 919 CCTTCTGGTTCTGCGCGGAGT 1001 PSGSARS 0.82811 920CCCAGCCACGCCAGAGCCAGC 1002 PSHARAS 0.520336 921 CCCGTCGGCACCAGAACCAGC1003 PVGTRTS 0.560759 922 AGAGACACCGTCAGCAGATAC 1004 RDTVSRY 0.535765923 AGAATCAGCACCCTCGGCCTC 1005 RISTLGL 0.595844 924AGGCTTGATAGGACTGGTTTG 1006 RLDRTGL 0.588542 925 AGACTCACCAACAGCAACCAA1007 RLTNSNQ 0.516161 926 CGTCAGCATCAGCTTCCTATG 1008 RQHQLPM 0.505918927 AGAACCGCCAACGCCCTCGGC 1009 RTANALG 0.802247 928CGGACGGATGTGCGGACGAAT 1010 RTDVRTN 0.682169 929 CGGACTACGGCTAATTCGCTT1011 RTTANSL 0.509233 930 AGCGGCGGCACCAGAGAAGGC 1012 SGGTREG 0.501512931 AGCATCAGAGCCCCCGTCAGC 1013 SIRAPVS 0.572426 932TCGATTTCTCCTCCTCGTACG 1014 SISPPRT 0.550578 933 TCGAAGGTTACGCCTCCTCTG1015 SKVTPPL 0.687491 934 AGCAGCAGACTCAGCGTCGGC 1016 SSRLSVG 0.637971935 AGCACCAGAAACGTCGTCGGC 1017 STRNVVG 0.532567 936TCGGTTCCTCCGAATAGGAAT 1018 SVPPNRN 0.508166 937 ACGGCTGCGCATGTTAGTTAT1019 TAAHVSY 0.520336 938 ACCAGAAGCGAAGTCATGAAA 1020 TRSEVMK 0.683884939 GTGGCGGGTCTGACTGTTCAG 1021 VAGLTVQ 0.581695 940GTTGGTTCTAGTAATACGTAT 1022 VGSSNTY 0.502229 941 GTTAAGACTGATCGGGTTCTG1023 VKTDRVL 0.51183 942 GTCAACAGCCACGAAAGAGCC 1024 VNSHERA 0.573043 943GTCCAAGTCGCCATGAGAGCC 1025 VQVAMRA 0.520831 944 GTCAGCATCAGCGTCATGGGC1026 VSISVMG 0.510987 945 GTGACGGGGGTTCGGATTGGG 1027 VTGVRIG 0.75699 946TGGTCTGATCCTAGTGATCTG 1028 WSDPSDL 0.59828 947 TACCTCACCAGCGGCGGCTAC1029 YLTSGGY 0.538789 948 TACCCCCACATGACCCACGAC 1030 YPHMTHD 0.655794949 TATGTTAATAGTGCTCCGAAG 1031 YVNSAPK 0.551681

The AAV capsids and AAV capsid proteins disclosed herein, in someembodiments, are isolated. In some instances, the AAV capsids and AAVcapsid proteins disclosed herein are isolated and purified. In addition,the AAV capsids and AAV capsid proteins disclosed herein, eitherisolated and purified, or not, may be formulated into a pharmaceuticalformulation, which in some cases, further comprises a pharmaceuticallyacceptable carrier.

B. Heterologous Nucleic Acids

Disclosed herein are therapeutic nucleic acids useful for the treatmentor prevention of a disease or condition, or symptom of the disease orcondition, disclosed herein. In some embodiments, the therapeuticnucleic acids encode a therapeutic gene expression product. Non-limitingexamples of gene expression products include proteins, polypeptides,peptides, enzymes, antibodies, antigen binding fragments, nucleic acid(RNA, DNA, antisense oligonucleotide, siRNA, and the like), and geneediting components, for use in the treatment, prophylaxis, and/oramelioration of the disease or disorder, or symptoms of the disease ordisorder. In some instances, the therapeutic nucleic acids are placed inan organism, cell, tissue or organ of a subject by way of a rAAV, suchas those disclosed herein.

Disclosed herein are rAAVs, each comprising a viral vector (e.g., asingle stranded DNA molecule (ssDNA)). In some instances, the viralvector comprises two inverted terminal repeat (ITR) sequences that areabout 145 bases each, flanking a transgene. In some embodiments, thetransgene comprises a therapeutic nucleic acid, and in some cases, apromoter in cis with the therapeutic nucleic acid in an open readingframe (ORF). The promoter is capable of initiating transcription oftherapeutic nucleic acid in the nucleus of the target cell. The ITRsequences can be from any AAV serotype. Non-limiting examples of AAVserotypes include AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, and AAV12. In some cases, an ITR is from AAV2. In somecases, an ITR is from AAV9.

Disclosed herein are transgenes that can comprise any number ofnucleotides. In some cases, a transgene can comprise less than about 100nucleotides. In some cases, a transgene can comprise at least about 100nucleotides. In some cases, a transgene can comprise at least about 200nucleotides. In some cases, a transgene can comprise at least about 300nucleotides. In some cases, a transgene can comprise at least about 400nucleotides. In some cases, a transgene can comprise at least about 500nucleotides. In some cases, a transgene can comprise at least about 1000nucleotides. In some cases, a transgene can comprise at least about 5000nucleotides. In some cases, a transgene can comprise at least about10,000 nucleotides. In some cases, a transgene can comprise at leastabout 20,000 nucleotides. In some cases, a transgene can comprise atleast about 30,000 nucleotides. In some cases, a transgene can compriseat least about 40,000 nucleotides. In some cases, a transgene cancomprise at least about 50,000 nucleotides. In some cases, a transgenecan comprise between about 500 and about 5000 nucleotides. In somecases, a transgene can comprise between about 5000 and about 10,000nucleotides. In any of the cases disclosed herein, the transgene cancomprise DNA, RNA, or a hybrid of DNA and RNA. In some cases, thetransgene can be single stranded. In some cases, the transgene can bedouble stranded.

Disclosed herein are transgenes useful for modulating the expression oractivity of a target gene or gene expression product thereof. In someinstances, the transgene is encapsidated by an rAAV capsid protein of anrAAV particle described herein. In some instances, the rAAV particle isdelivered to a subject to treat a disease or condition disclosed hereinin the subject. In some instances, the delivery is systemic (e.g.,intravenous, intranasal).

The transgenes disclosed herein are useful for expressing an endogenousgene at a level similar to that of a healthy or normal individual. Thisis particularly useful in the treatment of a disease or conditionrelated to the underexpression, or lack of expression, of a geneexpression product. In some embodiments, the transgenes disclosed hereinare useful for overexpressing an endogenous gene, such that anexpression level of the endogenous gene is above the expression level ofa healthy or normal individual. Additionally, transgenes can be used toexpress exogenous genes (e.g., active agent such as an antibody,peptide, nucleic acid, or gene editing components). In some embodiments,the therapeutic gene expression product is capable of altering,enhancing, increasing, or inducing the activity of one or moreendogenous biological processes in the cell. In some embodiments, thetransgenes disclosed herein are useful for reducing expressing anendogenous gene, example, a dominant negative gene. In some embodiments,the therapeutic gene expression product is capable of altering,inhibiting, reducing, preventing, eliminating, or impairing the activityof one or more endogenous biological processes in the cell. In someaspects, the increase of gene expression refers to an increase by atleast about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%.In one aspect, the protein product of the targeted gene may be increasedby at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and100%. In some aspects, the decrease of gene expression refers to anincrease by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95% and 100%. In one aspect, the protein product of the targeted genemay be decreased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%,85%, 90%, 95% and 100%.

When endogenous sequences (endogenous or part of a transgene) areexpressed with a transgene, the endogenous sequences can be full-lengthsequences (wild-type or mutant) or partial sequences. The endogenoussequences can be functional. Non-limiting examples of the function ofthese full length or partial sequences include increasing the serumhalf-life of the polypeptide expressed by a transgene (e.g., therapeuticgene) and/or acting as a carrier.

A transgene can be inserted into an endogenous gene such that all, someor none of the endogenous gene is expressed. For example, a transgene asdescribed herein can be inserted into an endogenous locus such that some(N-terminal and/or C-terminal to a transgene) or none of the endogenoussequences are expressed, for example as a fusion with a transgene. Inother cases, a transgene (e.g., with or without additional codingsequences of the endogenous gene) is integrated into any endogenouslocus, for example a safe-harbor locus. For example, a Frataxin (FXN)transgene can be inserted into an endogenous FXN gene. A transgene canbe inserted into any gene, e.g., the genes as described herein.

At least one advantage of the present disclosure is that virtually anytherapeutic nucleic acid may be used to express any therapeutic geneexpression product. In some instances, the therapeutic gene expressionproduct is a therapeutic protein or a peptide (e.g., antibody,antigen-binding fragment, peptide, or protein). In one embodiment theprotein encoded by the therapeutic nucleic acid is between 50-5000 aminoacids in length. In some embodiments the protein encoded is between50-2000 amino acids in length. In some embodiments the protein encodedis between 50-1000 amino acids in length. In some embodiments theprotein encoded is between 50-1500 amino acids in length. In someembodiments the protein encoded is between 50-800 amino acids in length.In some embodiments the protein encoded is between 50-600 amino acids inlength. In some embodiments the protein encoded is between 50-400 aminoacids in length. In some embodiments the protein encoded is between50-200 amino acids in length. In some embodiments the protein encoded isbetween 50-100 amino acids in length. In some embodiments the peptideencoded is between 4-50 amino acids in length. In some embodiments, theprotein encoded is a tetrapeptide, a pentapeptide, a hexapeptide, aheptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In someembodiments, the protein encoded comprises a peptide of 2-30 aminoacids, such as for example 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20amino acids. In some embodiments, the protein encoded comprises apeptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, ora peptide that is no longer than 50 amino acids, e.g. no longer than 35,30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.

Non-limiting examples of therapeutic protein or peptides include anadrenergic agonist, an anti-apoptosis factor, an apoptosis inhibitor, acytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, aglutamic acid decarboxylase, a glycoprotein, a growth factor, a growthfactor receptor, a hormone, a hormone receptor, an interferon, aninterleukin, an interleukin receptor, a kinase, a kinase inhibitor, anerve growth factor, a netrin, a neuroactive peptide, a neuroactivepeptide receptor, a neurogenic factor, a neurogenic factor receptor, aneuropilin, a neurotrophic factor, a neurotrophin, a neurotrophinreceptor, an N-methyl-D-aspartate antagonist, a plexin, a protease, aprotease inhibitor, a protein decarboxylase, a protein kinase, a proteinkinsase inhibitor, a proteolytic protein, a proteolytic proteininhibitor, a semaphoring, a semaphorin receptor, a serotonin transportprotein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, aserpin receptor, and a tumor suppressor. In certain embodiments, thetherapeutic protein or peptide is selected from the group consisting ofbrain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor(CNTF), macrophage colony-stimulating factor (CSF), epidermal growthfactor (EGF), fibroblast growth factor (FGF), gonadotropin,interferon-gamma (IFN), insulin-like growth factor 1 (IFG-1), nervegrowth factor (NGF), platelet-derived growth factor (PDGF), pigmentepithelium-derived factor (PEDF), transforming growth factor (TGF),transforming growth factor-beta (TGF-B), tumor necrosis factor (TNF),vascular endothelial growth factor (VEGF), prolactin, somatotropin,X-linked inhibitor of apoptosis protein 1 (XIAP1), interleukin 1 (IL-1),IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10, viralIL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18.

A therapeutic gene expression product can comprise gene editingcomponents. Non-limiting examples of gene editing components includethose required for CRISPR/Cas, artificial site-specific RNA endonuclease(ASRE), zinc finger endonuclease (ZFN), and transcription factor likeeffector nuclease (TALEN). In a non-limiting example, a subject havingHuntington's disease is identified. The subject is then systemicallyadministered a first amount of a rAAV encapsidating a viral vectorencoding ZFN engineered to represses the transcription of the Huntingtin(HTT) gene. In some instances, the route of administration isintravenous. The rAAV will include a modified AAV capsid protein thatincludes an amino acid sequence provided in any one of Tables 2-3, orFIG. 33, so as to allow proper targeting of the ZFN to the nervoussystem, while retargeting off-target organs, such as the liver. Ifneeded, the subject is administered a second or third dose of the rAAV,until a therapeutically effective amount of the ZFN is expressed in thesubject's nervous system. In another non-limiting example, a subjectwith cystic fibrosis is identified. The subject is then systemicallyadministered a first amount of a rAAV encapsidating a viral vectorencoding ZFN engineered to represses the transcription of the cysticfibrosis transmembrane conductance regulator (CFTR) gene. In someinstances, the route of administration is intranasal (e.g., intranasalspray). The rAAV will include a modified AAV capsid protein thatincludes an amino acid sequence provided in FIG. 33 or Tables 2-3, so asto allow proper targeting of the ZFN to the lung. If needed, the subjectis administered a second or third dose of the rAAV, until atherapeutically effective amount of the ZFN is expressed in thesubject's lung.

A therapeutic nucleic acid can comprise a non-protein coding gene e.g.,sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs (miRNAs),miRNA sponges or decoys, recombinase delivery for conditional genedeletion, conditional (recombinase-dependent) expression, includes thoserequired for the gene editing components described herein. Thenon-protein coding gene may also encode a tRNA, rRNA, tmRNA, piRNA,double stranded RNA, snRNA, snoRNA, and/or long non-coding RNA (IncRNA).In some cases, the non-protein coding gene can modulate the expressionor the activity of a target gene or gene expression product. Forexample, the RNAs described herein may be used to inhibit geneexpression in a target cell, for example, a cell in the central nervoussystem (CNS) or peripheral organ (e.g., lung). In some cases, inhibitionof gene expression refers to an inhibition by at least about 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In some cases, theprotein product of the targeted gene may be inhibited by at least about20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. The gene canbe either a wild type gene or a gene with at least one mutation. Thetargeted protein may be either a wild type protein or a protein with atleast one mutation.

A therapeutic nucleic acid can modulate the expression or activity of agene or gene expression product expressed from the gene that isimplicated in disease or disorder of the brain. For example, thetherapeutic nucleic acid, in some cases is a gene or a modified versionof the gene described herein. In another example, the therapeuticnucleic acid comprises an effector gene expression product such as agene editing component specific to target a gene therein. Non-limitedexamples of genes include Sarcoglycan Alpha (SGCA), glutamic aciddecarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2gene, Nerve Growth Factor (NGF), glial cell derived neurotrophicfactor(GDNF), Neurturin, Survival Of Motor Neuron 1, Telomeric (SMN1),β-Glucocerebrosidase (GCase), Frataxin (FXN), Huntingtin (HTN),methyl-CpG binding protein 2 (MECP2), peroxisomal biogenesis factor(PEX), progranulin (GRN), an antitubulin agent, copper-zinc superoxidedismutase (SOD1), Glucosylceramidase Beta (GBA), NPC IntracellularCholesterol Transporter 1 (NPC1), and NPS3. In some embodiments, theperoxisomal biogenesis factor (PEX) is selected from the groupconsisting of PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11(3,PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. In some instances, thegene or gene expression product is inhibited. In some instances, thegene or gene expression product is enhanced.

A therapeutic nucleic acid modulates expression or activity of a gene orgene expression product expressed from the gene that is implicated indisease or disorder of a particular organ (e.g., lung, heart, liver,muscle, eye). Non-limited examples of genes include Cystic FibrosisTransmembrane Conductance Regulator (CFTR), Factor X (FIX), RPE65,Retinoid Isomerohydrolase (RPE65), Sarcoglycan Alpha (SGCA), andsarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a). In some embodiments,the therapeutic gene expression product is of human, murine, avian,porcine, bovine, ovine, feline, canine, equine, epine, caprine, lupineor primate origin. In some instances, the gene or gene expressionproduct is inhibited. In some instances, the gene or gene expressionproduct is enhanced.

C. AAV Vectors

Disclosed herein are adeno-associated virus (AAV) vectors comprisinggenetic information. AAV vectors described herein are useful for theassembly of a rAAV and viral packaging of a heterologous nucleic acid.In addition, an AAV vector may encode a transgene comprising theheterologous nucleic acid. In some instances, the AAV vector is from anAAV serotypes selected from the group consisting of AV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In someinstances, the AAV vector is selected from a modified AAV serotypeselected from the group consisting of AAV-PHP.B, AAV-PHP.eB, andAAV-PHP.S.

An AAV vector can comprise a transgene, which in some cases encodes aheterologous gene expression product (e.g., therapeutic gene expressionproduct, recombinant capsid protein, and the like). The transgene is incis with two inverted terminal repeats (ITRs) flanking the transgene.The transgene may comprise a therapeutic nucleic acid encoding atherapeutic gene expression product. Due to the limited packagingcapacity of the rAAV (˜2.5 kB), in some cases, a longer transgene may besplit between two AAV vectors, the first with 3′ splice donor and thesecond with a 5′ splice acceptor. Upon co-infection of a cell,concatemers form, which are spliced together to express a full-lengthtransgene.

A transgene is generally inserted so that its expression is driven bythe endogenous promoter at the integration site, namely the promoterthat drives expression of the endogenous gene into which a transgene isinserted. In some instances, a transgene comprises a promoter and/orenhancer, for example a constitutive promoter or an inducible ortissue/cell specific promoter. As a non-limiting example, the promotermay be CMV promoter, a CMV-(3-Actin-intron-(3-Globin hybrid promoter(CAG), CBA promoter, FRDA or FXN promoter, UBC promoter, GUSB promoter,NSE promoter, Synapsin promoter, MeCP2 promoter, GFAP promoter, H1promoter, U6 promoter, NFL promoter, NFH promoter, SCN8A promoter, orPGK promoter. As a non-limiting example, promoters can betissue-specific expression elements include, but are not limited to,human elongation factor 1a-subunit (EF1a), immediate-earlycytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, the(3 glucuronidase (GUSB), and ubiquitin C (UBC). The transgene mayinclude a tissue-specific expression elements for neurons such as, butnot limited to, neuron-specific enolase (NSE), platelet-derived growthfactor (PDGF), platelet-derived growth factor B-chain (PDGF-(3), thesynapsin (Syn), the methyl-CpG binding protein 2 (MeCP2),Ca2+/calmodulin-dependent protein kinase II (CaMKII), metabotropicglutamate receptor 2 (mGluR2), NFL, NFH, np32, PPE, Enk and EAAT2promoters. The transgene may comprise a tissue-specific expressionelement for astrocytes such as, but not limited to, the glial fibrillaryacidic protein (GFAP) and EAAT2 promoters. The transgene may comprisetissue-specific expression elements for oligodendrocytes such as, butnot limited to, the myelin basic protein (MBP) promoter.

In some embodiments, the promoter is less than 1 kb. The promoter mayhave a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800 or more than 800. The promotermay have a length between 200-300, 200-400, 200-500, 200-600, 200-700,200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600,400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or700-800. The promoter may provide expression of the therapeutic geneexpression product for a period of time in targeted tissues such as, butnot limited to, the central nervous system and peripheral organs (e.g.,lung). Expression of the therapeutic gene expression product may be fora period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years,12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years, ormore than 65 years. Expression of the payload may be for 1-5 hours, 1-12hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months,1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months,6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8years or 5-10 years or 10-15 years, or 15-20 years, or 20-25 years, or25-30 years, or 30-35 years, or 35-40 years, or 40-45 years, or 45-50years, or 50-55 years, or 55-60 years, or 60-65 years.

An AAV vector can comprise a genome of a helper virus. Helper virusproteins are required for the assembly of a recombinant AAV (rAAV), andpackaging of a transgene containing a heterologous nucleic acid into therAAV. The helper virus genes are adenovirus genes E4, E2a and VA, thatwhen expressed in the cell, assist with AAV replication. In someembodiments, an AAV vector comprises E2. In some embodiments, an AAVvector comprises E4. In some embodiments, an AAV vector comprises VA. Insome instances, the AAV vector comprises one of helper virus proteins,or any combination.

An AAV vector can comprise a viral genome comprising a nucleic acidencoding the recombinant AAV (rAAV) capsid protein described herein. Theviral genome can comprise a

Replication (Rep) gene encoding a Rep protein, and Capsid (Cap) geneencoding an AAP protein in the first open reading frame (ORF1) or a Capprotein in the second open reading frame (ORF2). The Rep protein isselected from the group consisting of Rep78, Rep68, Rep52, and Rep40. Insome instances, the Cap gene is modified encoding a modified AAV capsidprotein described herein. A wild-type Cap gene encodes three proteins,VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 ismodified. In some cases, VP3 is modified. In some cases, all threeVP1-VP3 are modified. The AAV vector can comprise nucleic acids encodingwild-type Rep78, Rep68, Rep52, Rep40 and AAP proteins.

Disclosed herein are AAV vectors comprising any one of SEQ ID NOS:10-434, 860-863, 868-949, 1068-5661, 14841-14880, and 14961-15053 whichare the DNA sequences encoding modified portions of AAV capsid proteinsof the present disclosure. In some instances, the AAV vector comprises anucleic acid sequences provided in any one of SEQ ID NOS: 10-434 and868-949, encoding 7-mer modified AAV capsid protein portions. The AAVvector of the present disclosure can comprise the VP1 Cap gene comprisesany one of SEQ ID NOS: 6-9 provided in Table 5. An AAV vector cancomprise 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of any one of SEQ ID NOS: 6-9.

In some instances, the AAV9 VP1 gene provided in SEQ ID NO: 6, which isprovided in Table 5, is modified to include any one of SEQ ID NOS:10-434, 860-863, 868-949, 1068-5661, 14841-14880, and 14961-15053. Insome instances, the AAV-PHP.eB VP1 (SEQ ID NO: 9), which is alsoprovided in Table 5 is modified to include any one of SEQ ID NOS:10-434, 860-863, 868-949, 1068-5661, 14841-14880, and 14961-15053. TheAAV vector described herein may be used to produce a variant AAV capsidby the methods described herein.

TABLE 5 VP1 Capsid Protein Nucleic Acid Sequences SEQ ID NO: IdentifierSequence 6 AAV9 ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAA >AY530579.1CCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTG Adeno-GAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAA associatedCGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACC virus 9CGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCA isolateGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGC hu.14 capsidAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCAC protein VP1GCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTC (cap) gene,TTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAA completeAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCT cdsAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA 7 AAV-ATGGCTGCCG ATGGTTATCT TCCAGATTGG CTCGAGGACA PHP.BACCTTAGTGAAGGAATTCGC GAGTGGTGGG CTTTGAAACCTGGAGCCCCT CAACCCAAGGCAAATCAACA ACATCAAGACAACGCTCGAG GTCTTGTGCT TCCGGGTTACAAATACCTTGGACCCGGCAA CGGACTCGAC AAGGGGGAGC GGTCAACGCAGCAGACGCG GCGGCCCTCG AGCACGACAA GCCTACGACCAGCAGCTCAAGGCCGGAGA CAACCCGTAC CTCAAGTACAACCACGCCGA CGCCGAGTTCCAGGAGCGGC TCAAAGAAGATACGTCTTTT GGGGGCAACC TCGGGCGAGCAGTCTTCCAGGCCAAAAAGA GGCTTCTTGA ACCTCTTGGT CTGGTTGAGGAAGCGGCTAA GACGGCTCCT GGAAAGAAGAGGCCTGTAGA GCAGTCTCCTCAGGAACCGG ACTCCTCCGCGGGTATTGGC AAATCGGGTG CACAGCCCGCTAAAAAGAGACTCAATTTCG GTCAGACTGG CGACACAGAGTCAGTCCCAGACCCTCAACC AATCGGAGAA CCTCCCGCAGCCCCCTCAGG TGTGGGATCT CTTACAATGG CTTCAGGTGGTGGCGCACCA GTGGCAGACA ATAACGAAGGTGCCGATGGAGTGGGTAGTT CCTCGGGAAA TTGGCATTGCGATTCCCAATGGCTGGGGGA CAGAGTCATC ACCACCAGCACCCGAACCTG GGCCCTGCCCACCTACAACA ATCACCTCTACAAGCAAATC TCCAACAGCA CATCTGGAGGATCTTCAAAT GACAACGCCT ACTTCGGCTA CAGCACCCCCTGGGGGTATTTTGACTTCAA CAGATTCCAC TGCCACTTCTCACCACGTGA CTGGCAGCGACTCATCAACA ACAACTGGGGATTCCGGCCT AAGCGACTCA ACTTCAAGCTCTTCAACATTCAGGTCAAAG AGGTTACGGA CAACAATGGAGTCAAGACCA TCGCCAATAA CCTTACCAGC ACGGTCCAGGTCTTCACGGA CTCAGACTATCAGCTCCCGT ACGTGCTCGGGTCGGCTCAC GAGGGCTGCC TCCCGCCGTTCCCAGCGGACGTTTTCATGA TTCCTCAGTA CGGGTATCTGACGCTTAATGATGGAAGCCA GGCCGTGGGT CGTTCGTCCTTTTACTGCCT GGAATATTTC CCGTCGCAAA TGCTAAGAACGGGTAACAAC TTCCAGTTCA GCTACGAGTTTGAGAACGTACCTTTCCATA GCAGCTACGC TCACAGCCAAAGCCTGGACCGACTAATGAA TCCACTCATC GACCAATACTTGTACTATCT CTCTAGAACT ATTAACGGTT CTGGACAGAATCAACAAACG CTAAAATTCA GTGTGGCCGGACCCAGCAAC ATGGCTGTCC AGGGAAGAAA CTACATACCTGGACCCAGCTACCGACAACA ACGTGTCTCA ACCACTGTGACTCAAAACAA CAACAGCGAATTTGCTTGGC CTGGAGCTTCTTCTTGGGCT CTCAATGGAC GTAATAGCTT GATGAATCCTGGACCTGCTA TGGCCAGCCA CAAAGAAGGAGAGGACCGTTTCTTTCCTTT GTCTGGATCT TTAATTTTTGGCAAACAAGG TACCGGCAGAGACAACGTGG ATGCGGACAA AGTCATGATA ACCAACGAAGAAGAAATTAAAACTACTAAC CCGGTAGCAA CGGAGTCCTATGGACAAGTG GCCACAAACCACCAGAGTGC CCAAACTTTGGCGGTGCCTT TTAAGGCACA GGCGCAGACCGGTTGGGTTCAAAACCAAGG AATACTTCCG GGTATGGTTTGGCAGGACAGAGATGTGTAC CTGCAAGGAC CCATTTGGGCCAAAATTCCT CACACGGACG GCAACTTTCA CCCTTCTCCGCTGATGGGAG GGTTTGGAAT GAAGCACCCGCCTCCTCAGATCCTCATCAA AAACACACCT GTACCTGCGGATCCTCCAACGGCCTTCAAC AAGGACAAGC TGAACTCTTTCATCACCCAG TATTCTACTGGCCAAGTCAG CGTGGAGATCGAGTGGGAGC TGCAGAAGGA AAACAGCAAGCGCTGGAACC CGGAGATCCA GTACACTTCC AACTATTACAAGTCTAATAATGTTGAATTT GCTGTTAATA CTGAAGGTGTATATAGTGAA CCCCGCCCCATTGGCACCAG ATACCTGACT CGTAATCTGT AA 8 AAV-ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAA PHP.SCCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAACAGGCGGTTAGGACGTCTTTGGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGA CTCGTAATCTGTAA 9 AAV-ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAA PHP.eBCCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCTAGAACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGTACCGGCAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGATGGGACTTTGGCGGTGCCTTTTAAGGCACAGGCGCAGACCGGTTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGA CTCGTAATCTGTAA

II. METHODS

A. Methods of Producing AAVs

Disclosed herein are methods of identifying a recombinant AAV (rAAV),such as those disclosed herein. The AAV peptide specific to target invivo environments are identified using multiplexed Crerecombination-based AAV targeted evolution (M-CREATE). FIG. 1 provides aworkflow using M-CREATE. M-CREATE uses an rAAV capsid genome(rAAV-Cap-in-cis-lox or rAAV-ACap-in-cis-lox2 as described in Example 2below) that couples a full-length AAV Cap gene, controlled by regulatoryelements from the AAV Rep gene, with a Cre-invertible switch. TherAAV-ACap-in-cis-lox2 backbone has a bi-directional polyA flanked by twoLox sites (lox71 and lox66). The ACap backbone is nonfunctional as it ismissing a portion of the capsid gene. Upon insertion of a libraryfragment with mutagenesis at a specific site into the capsid, it thenbecomes a fully functional vector. In a cell expressing the Crerecombinase, the Cre-Lox recombination facilitates inversion of thepolyA in addition to flipping the Lox sites to Lox72 and LoxP. (SeeFIGS. 1 and 36) The randomized 7-mer and 11-mers disclosed herein aregenerated using PCR, which are then inserted into therAAV-ACap-in-cis-lox to generate virus libraries with randomizedinsertions or substitutions in the capsid protein sequence (e.g., at theAA588 589). (FIG. 1). In a first round of in vivo selection, the viruslibraries are injected into the blood stream (e.g., intravenously) oftransgenic animals expressing Cre recombinase. Tissues obtained from thetransgenic animals following injection in in vivo environment (e.g.,brain/liver). The inverted reporter expression cassette is detectedusing selective amplification expression of the reporter gene expressionproduct. The rAAVs in the tissues are isolated and the viral genomearound the insertion site is sequenced and aligned with an AAV9 templateDNA fragment. The 7-mers and 11-mers that were recovered were enrichedin the target in vivo environment, and were cloned into anotherrAAV-ACap-in-cis-lox2 backbone, and another round of in vivo selectionis performed. The 7-mers and 11-mers enriched in the target in vivoenvironment, and negatively enriched in an off-target in vivoenvironment are sequenced using suitable methods, such as nextgeneration sequencing. FIGS. 33-35 provide DNA sequences identifiedusing the methods provided herein.

Methods comprise providing a rAAV genome comprising an AAV capsid gene,and a recognition sequence for a Cre recombinase. In some cases, therAAV genome has two recognition sequences for Cre recombinase that flanka reporter expression cassette. The recognition sequences for Crerecombinase (e.g., LoxP) are oriented such that an inversion of thereporter cassette is facilitated in the presence of Cre recombinase in acell. Methods comprise transfecting a population of cells expressing theCre recombinase with the rAAV genome. The Cre recombinase induces aninversion (e.g., “flip” of the reporter gene into a genome of thetransgenic animal). In some cases, the rate of inversion (e.g., a levelof expression of the reporter gene in a target cell) may be measuredusing any suitable method, such as quantitative polymerase chainreaction, or immunohistochemistry. The level of expression of thereporter gene is compared to a reference value, which in some cases isthe rate of inversion by a reference AAV (e.g., AAV9). Methods disclosedherein provide that an increase in the rate of inversion is detected ina target in vivo environment, as compared to the rate of inversion of areference AAV. In some cases, a decrease in a rate of inversion isdetected in an off-target cell, as compared to the rate of inversion ofa reference AAV. The rAAV genome recovered using the methods describedherein encodes for an AAV capsid particle (e.g., capsid protein, capsid)with an increased specificity for the target cell, and a decreasedspecificity for the off-target cell.

Disclosed herein are methods of producing a rAAV disclosed herein. Insome instances, all elements that are required for AAV production intarget cell (e.g., HEK293cells) are transiently transfected into thetarget cell using suitable methods known in the art. For example, therAAV of the present disclosure can be product by co-transfecting threeplasmid vectors, a first vector with ITR-containing plasmid carrying thetransgene (e.g., therapeutic nucleic acid), a second vector that carriesthe AAV Rep and Cap genes; and (3), a third vector that provides thehelper genes isolated from adenovirus. The methods described hereingenerate high-titer AAV vectors that are free of adenovirus. The Capgene disclosed here comprises any one of SEQ ID NOS: 10-434, 860-863,868-949, 1068-5661, 14841-14880, and 14961-15053 which are DNA sequencesencoding the modified AAV capsid protein portions of the presentdisclosure. In some cases, rAAVs of the present disclosure are generatedusing the methods described in Challis, R. C. et al. Systemic AAVvectors for widespread and targeted gene delivery in rodents. Nat.Protoc. 14, 379 (2019), which is hereby incorporated by reference in itsentirety. Briefly, triple transfection of HEK293T cells (ATCC) usingpolyethylenimine (PEI) is performed, viruses are collected after 120hours from both cell lysates and media, and purified over iodixanol.

Disclosed herein, are methods comprising: (a) introducing into a cell anucleic acid comprising: (1) a first nucleic acid sequence encoding atherapeutic gene expression product; (2) a second nucleic acid sequenceencoding viral genome components comprising: (i) a Replication (Rep)gene encoding a Rep protein; and (ii) a modified capsid (Cap) geneencoding a modified AAV capsid protein described herein, and (3) a thirdnucleic acid sequence encoding a genome of an AAV helper virus; and (b)assembling a recombinant AAV (rAAV) capsid encapsidating the firstnucleic acid, the rAAV capsid comprising a tropism with an increasedspecificity for a target in vivo environment in a subject and adecreased specificity for an off-target in vivo environment, relative toa tropism of a corresponding parental AAV capsid protein. In someinstances, the methods further comprise packing the first nucleic acidsequence encoding the therapeutic gene expression product such that itbecomes encapsidated by the modified AAV capsid protein. In someembodiments, the rAAV particles are isolated, concentrated, and purifiedusing suitable viral purification methods, such as those describedherein.

In a non-limiting example, the rAAVs are generated by tripletransfection of precursor cells (e.g., HEK293T) cells using a standardtransfection protocol (e.g., PEI). Viral particles are harvested fromthe media after a period of time (e.g., 72 h post transfection) and fromthe cells and media at a later point in time (e.g., 120 h posttransfection). Virus present in the media is concentrated byprecipitation with 8% poly(ethylene glycol) and 500 mM sodium chlorideand the precipitated virus is added to the lysates prepared from thecollected cells. The viruses are purified over iodixanol (Optiprep,Sigma) step gradients (15%, 25%, 40% and 60%). Viruses are concentratedand formulated in PBS. Virus titers are determined by measuring thenumber of DNaseI-resistant vector genome copies (VGs) using qPCR and thelinearized genome plasmid as a control.

The Rep protein can be selected from the group consisting of Rep78,Rep68, Rep52, and Rep40. The genome of the AAV helper virus comprises anAAV helper gene selected from the group consisting of E2, E4, and VA.The second nucleic acid and the first nucleic acid can be in trans. Thesecond nucleic acid and the first nucleic acid can be in cis.

The cell can be selected from a group consisting of a human, a primate,a murine, a feline, a canine, a porcine, an ovine, a bovine, an equine,an epine, a caprine and a lupine host cell. In some instances, the cellis a progenitor or precursor cell, such as a stem cell. In someinstances, the stem cell is a mesenchymal cell, embryonic stem cell,induced pluripotent stem cell (iPSC), fibroblast or other tissuespecific stem cell. The cell can be immortalized. In some cases, theimmortalized cell is a HEK293cell. In some instances, the cell is adifferentiated cell. Based on the disclosure provided, it is expectedthat this system can be used in conjunction with any transgenic lineexpressing a recombinase in the target cell type of interest to developAAV capsids that more efficiently transduce that target cell population.

B. Methods of rAAV-Mediated Delivery of a Heterologous Nucleic Acid

Disclosed herein are methods of delivering a heterologous nucleic acid(e.g., therapeutic nucleic acid or transgene disclosed herein) tosubject in need thereof. The transgene may be encapsidated by arecombinant AAV (rAAV) capsid protein or rAAV particle such as thosedescribed herein.

Methods may be ex vivo, e.g., scientific research purposes or forproducing adoptive cellular therapies. The subject may be a humanprimary cell or a mature cell, or cell line. The subject may be a cellfrom a monkey, hamster, or mouse. In either case, delivery may includecontacting the composition with the cell or cell line.

Methods may be in vivo, e.g., treating a disease or a condition in asubject in need thereof. In some instances, the subject may be mammal,such as a human or non-human primate, in which case delivery of thecomposition may comprise administering the composition to the subject.In some embodiments, delivery of the heterologous nucleic acid comprisesadministering to the subject the composition using any one of the routesof administration described herein.

In some embodiments, methods of increasing transduction of aheterologous nucleic acid in a target in vivo environment comprisedelivering a rAAV particle described herein, the rAAV engineered to havean increased transduction efficiency in a target in vivo environment(e.g., tissue or cell type). In some instances, the increasedtransduction efficiency comprises a 1-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold,21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold,29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold,37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold,45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 75-fold, or100-fold increase, or more, relative to a reference AAV. In someinstances, the increased transduction efficiency is at least 30-fold. Insome instances, the increased transduction efficiency is at least40-fold. In some instances, the increased transduction efficiency is atleast 50-fold. In some instances, the increased transduction efficiencyis at least 60-fold. In some instances, the increased transductionefficiency is at least 80-fold. In some instances, the increasedtransduction efficiency is at least 90-fold. In some instances, theincreased transduction efficiency is at least 100-fold.

Methods of delivering a heterologous nucleic acid to a target in vivoenvironment are also provided comprising delivering the rAAV particledescribed herein that has been engineered to have an increasedspecificity in a target in vivo environment (e.g., tissue or cell type),as compared to a reference AAV. Methods, in some cases, comprisedetecting whether a rAAV possesses more specificity for a target in vivoenvironment than a reference AAV, includes measuring a level of geneexpression product (e.g., RNA or protein) expressed from theheterologous nucleic acid encapsidated by the rAAV in a tissue sampleobtained from the target in vivo environment in a subject; and comparingthe measured level to a control level (such as, for e.g., the geneexpression product expressed from a heterologous nucleic acidencapsidated by a reference AAV (e.g., AAV9)). Suitable methods formeasuring expression of a gene expression product luciferase reporterassay and quantitative polymerase chain reaction (qPCR).

In some instances, the reference AAV has a serotype selected from thegroup consisting of AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, AAV12, or variant thereof. For example, thereference AAV can have a serotype selected from the group consisting ofAAV-PHP.B, AAV-PHP.eB, and AAV-PHP.S.

Delivery to the CNS

Provided herein are methods of delivering a heterologous nucleic acid toa target in vivo environment comprising delivering a composition to thetarget in vivo environment selected from the group consisting of acentral nervous system (CNS) in a subject, the composition comprising arAAV particle with a rAAV capsid protein, the rAAV capsid proteinencapsidating a viral vector encoding a heterologous nucleic acid (e.g.,therapeutic nucleic acid). In some embodiments, the rAAV particleencapsidating the heterologous nucleic acid comprises a rAAV capsidprotein engineered with an increased specificity and, in some cases,transduction efficiency when measured in the CNS or the PNS of thesubject, even when administered to the subject systemically, as comparedto a reference AAV.

Methods comprise delivering a rAAV particle comprising an rAAV capsidprotein with increased specificity and/or transduction efficiency whenmeasured in the CNS in the subject, as compared to a reference AAV(e.g., AAV9). In some embodiments, delivery is systemic. Alternatively,delivery is direct (e.g., into the affected area of the CNS).

The rAAV capsid protein may comprise a substitution of at least one,two, three, four, five, six, seven, eight, nine, ten, or eleven aminoacids provided in an amino acid sequence provided in any one of Tables2-3, in an amino acid sequence of a parental AAV. In some instances, X1is selected from the group consisting of A, E, D, G, R, S and T. In someinstances, the insertion further comprises two amino acids, wherein X2is selected from the group consisting of A, G, I, L, M, N, Q, R, T, andY. In some instances, the insertion further comprises three amino acids,wherein X3 is selected from the group consisting of E, K, L, T, and Q.In some instances, the insertion further comprises at least four aminoacids, wherein X1 is selected from the group consisting of A, E, D, G,R, S and T, X2 is selected from the group consisting of A, G, I, L, M,N, Q, R, T, and Y, X3 is selected from the group consisting of E, K, L,T, and Q, and X4 is selected from the group consisting of G, I, K, L, M,R, T, and V. In some instances, the insertion further comprises fiveamino acids wherein X5 is selected from the group consisting of A, D, G,P, L, Q, and V. In some instances, the insertion further comprises atleast six amino acids, wherein X6 is selected from the group consistingof F, K, L, N, P, Q, S, and V. In some instances, the insertion furthercomprises at least seven amino acids, wherein X7 is selected from thegroup consisting of I, K, L, P, S, and V.

Disclosed herein are methods comprising delivering a rAAV particleencapsidating a heterologous nucleic acid to a CNS in a subject, therAAV particle comprising (i) an increased specificity and/ortransduction efficiency of the heterologous nucleic acid for the CNS,wherein the rAAV particle has an rAAV capsid protein comprising aninsertion of at least or about three, four, five, six, or seven aminoacids of an amino acid sequence TALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, orRYQGDSV, or any amino acid sequence provided in Tables 2-3 or FIG. 33,at an amino acid position 588_589 in a parental AAV capsid protein. Insome embodiments, the delivering is systemic. In some embodiments, thedelivery is direct (e.g., injected into the in vivo environment). Insome embodiments, the parental AAV capsid protein is AAV9 capsid protein(for e.g., provided in SEQ ID NO: 1). In some embodiments, delivery ismore specific than a delivery of the heterologous nucleic acid by areference AAV, e.g., AAV9. In some embodiments, the delivery is systemic(e.g., intravenous). In some embodiments, the subject is a mammal. Insome embodiments, the subject is a human.

Delivery to the Liver

In some cases, the methods of delivering a heterologous nucleic acidcomprise delivering to a target in vivo environment in a subject acomposition, the composition comprising a rAAV particle with a rAAVcapsid protein, the rAAV capsid protein encapsidating a viral vectorencoding a heterologous nucleic acid (e.g., therapeutic nucleic acid).In some cases, the target in vivo environment is the liver. In someembodiments, the rAAV particle encapsidating the heterologous nucleicacid comprises a rAAV capsid protein engineered with an increasedspecificity and, in some cases, transduction efficiency when measured inthe target in vivo environment of the subject, even when administered tothe subject systemically.

In some embodiments, methods comprise delivering a rAAV particlecomprising an rAAV capsid protein with increased specificity and/ortransduction efficiency of the heterologous nucleic acid for the liverin the subject, as compared to a reference AAV (e.g., AAV9). In someembodiments, rAAVs optimized for targeting the liver have amino acidsequences that comprise an amino acid sequence provided in SEQ ID NOS:950-1031 and 15054-15146 (FIG. 35).

The rAAV capsid protein suitable for delivery of the heterologousnucleic acid to the liver can comprise an insertion of at least oneamino acid in a parental AAV capsid protein. In some instances, theinsertion comprises at least one, two, three, four, five, six, seven,eight, nine, ten, or eleven amino acids provided in an amino acidsequence provided in Table 4, or FIG. 35.

Disclosed herein are methods comprising delivering a rAAV particleencapsidating a heterologous nucleic acid to the target in vivoenvironment selected from the group consisting of the liver in asubject, the rAAV particle comprising an increased specificity and/ortransduction efficiency of the heterologous nucleic acid for the targetin vivo environment, wherein the rAAV particle has an rAAV capsidprotein comprising an insertion of at least or about three, four, five,six, seven, eight, nine, ten, or eleven amino acids of an amino acidsequence provided in Table 4, or FIG. 35. In some embodiments, deliveryis more specific than a delivery of the heterologous nucleic acid by areference AAV, e.g., AAV9. In some embodiments, methods further comprisereducing or ablating delivery of the heterologous nucleic acid in anoff-target in vivo environment, such as the liver, compared to areference AAV. In some embodiments, delivery is characterized by anincrease in efficiency of transduction (e.g., of the heterologousnucleic acid) in the target in vivo environment than a transductionefficiency in the target in vivo environment of the reference AAV. Insome embodiments, the delivery is systemic (e.g., intravenous). In someembodiments, the subject is a mammal. In some cases, the mammal is ahuman.

C. Methods of Treatment

Disclosed herein are methods of treating a disease or condition, or asymptom of the disease or condition, in a subject, comprisingadministrating of therapeutically effective amount of one or morecompositions (e.g., rAAV particle, AAV vector, pharmaceuticalcomposition) disclosed herein to the subject. In some embodiments, thecomposition is a rAAV capsid protein described herein. In someembodiments, the composition is an isolated and purified rAAV capsidprotein described herein. In some embodiments, the rAAV particleencapsidates an AAV vector comprising a transgene (e.g., therapeuticnucleic acid). In some embodiments, the composition is a rAAV capsidprotein described herein conjugated with a therapeutic agent disclosedherein. In some embodiments, the composition is a pharmaceuticalcomposition comprising the rAAV particle and a pharmaceuticallyacceptable carrier. In some embodiments, the one or more compositionsare administered to the subject alone (e.g., standalone therapy). Insome embodiments, the one or more compositions are administered incombination with an additional agent. In some embodiments, thecomposition is a first-line therapy for the disease or condition. Insome embodiments, the composition is a second-line, third-line, orfourth-line therapy, for the disease or condition.

Provided herein are methods of treating a disease or a condition, or asymptom of the disease or condition, in a subject, comprising: (a)diagnosing a subject with a disease or a condition affecting a target invivo environment; and (b) treating the disease or the condition byadministering to the subject a therapeutically effective amount of acomposition disclosed herein (e.g., rAAV particle, AAV vector,pharmaceutical composition), wherein the composition is engineered withan increased specificity for the target in vivo environment.

Disclosed herein are methods of treating a disease or a condition, or asymptom of the disease or the condition, afflicting a target in vivoenvironment in a subject comprising: (a) administering to the subject acomposition (e.g., rAAV particle, AAV vector, pharmaceuticalcomposition); and (b) expressing the therapeutic nucleic acid into atarget in vivo environment in the subject with an increased specificityand/or transduction efficiency, as compared to a reference AAV. In somecases, the reference AAV is AAV9, or a variant thereof.

Methods of treating a disease or condition affecting the central nervoussystem (CNS) comprise administering a rAAV particle to a CNS in asubject, the rAAV particle comprising an rAAV capsid protein comprisingan insertion of at least or about three, four, five, six, seven, eight,nine, ten, or eleven amino acids of an amino acid sequence TALKPFL,TTLKPFL, TLQIPFK, TMQKPFI, RYQGDSV, or TTLKPFS, or any amino acidsequence provided in Tables 2-3, or FIG. 33, at an amino acid position588_589 in a parental AAV capsid protein. In some cases, the insertionis not TLAVPFK, KFPVALT, SVSKPFL, FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, orYTLSQGW. In some embodiments, the parental AAV capsid protein is AAV9capsid protein (for e.g., provided in SEQ ID NO: 1. In some instances,the parental AAV capsid protein comprises an amino acid sequence that isat least 95%, 96%, 96.1, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%,96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%,97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%,98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9%, or 100.0% identical to SEQ ID NO: 1. In someembodiments, delivery is more specific than a delivery of theheterologous nucleic acid by a reference AAV, e.g., AAV9. In someembodiments, the delivery is systemic (e.g., intravenous). In someembodiments, the subject is a human or a non-human primate.

Methods of treating a disease or a condition afflicting a target in vivoenvironment comprising a liver comprise administering a rAAV particle tothe target in vivo environment in a subject, the rAAV particlecomprising an rAAV capsid protein comprising a substitution of at leastor about three, four, five, six, seven, eight, nine, ten, or eleven,amino acids of an amino acid sequence provided in any one of SEQ ID NOS:950-1031 and 15054-15146 (FIG. 35). In some embodiments, methodscomprise delivering a rAAV particle comprising an rAAV capsid proteinwith increased specificity for the liver in the subject, as compared toa reference AAV (e.g., AAV9). In some embodiments, rAAVs optimized fortargeting the liver have amino acid sequences comprising an amino acidsequence KAYSVQV, PSGSARS, and RTANALG at an amino acid position 588_589in a parental AAV capsid protein. In some embodiments, the parental AAVcapsid protein is AAV9 capsid protein (for e.g., provided in SEQ ID NO:1). In some instances, the parental AAV capsid protein comprises anamino acid sequence that is at least 95%, 96%, 96.1, 96.2%, 96.3%,96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%,97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%,98.4%, 98.5%, 98.6%, 98.7%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100.0% identical toSEQ ID NO: 1. In some embodiments, delivery is more specific than adelivery of the heterologous nucleic acid by a reference AAV, e.g.,AAV9. In some embodiments, the delivery is systemic (e.g., intravenousor intranasal). In some embodiments, the subject is a human or anon-human primate.

Also provided are methods of modulating a target gene expressionproduct, the methods comprising administering to a subject in needthereof a composition (e.g., rAAV particle, AAV vector, pharmaceuticalcomposition) disclosed herein. For example, methods provided hereincomprise administering to a subject a rAAV with a rAAV capsid proteinencapsidating a viral vector comprising a heterologous nucleic acid thatmodulates the expression or the activity of the target gene expressionproduct. In some embodiments, the disease or the condition ischaracterized by an increased or enhanced expression or activity of agene or gene expression product thereof, as compared to a normalindividual. In some cases, administering the therapeutically effectiveamount of the composition restores the expression or the activity of thegene or gene expression product thereof to a level that is typical in anormal individual. The term “normal individual” refers to an individualthat is not afflicted with the disease or the condition characterized bythe variation in expression or activity of the gene or gene expressionproduct thereof.

Non-limiting examples of genes involved in central nervous system (CNS)diseases or disorders include Sarcoglycan Alpha (SGCA), glutamic aciddecarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2gene, Nerve Growth Factor (NGF), glial cell derived neurotrophic factor(GDNF), Neurturin, Survival Of Motor Neuron 1, Telomeric (SMN1),β-Glucocerebrosidase (GCase), Frataxin (FXN), Huntingtin (HTN),methyl-CpG binding protein 2 (MECP2), peroxisomal biogenesis factor(PEX), progranulin (GRN), an antitubulin agent, copper-zinc superoxidedismutase (SOD1), Glucosylceramidase Beta (GBA), NPC IntracellularCholesterol Transporter 1 (NPC1), and NPS3. In some embodiments, theperoxisomal biogenesis factor (PEX) is selected from the groupconsisting of PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11(3,PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. Non-limiting examples ofgenes implicated in disease or disorder of a particular organ (e.g.,lung, heart, liver, muscle, eye) include Cystic Fibrosis TransmembraneConductance Regulator (CFTR), Factor X (FIX), RPE65, RetinoidIsomerohydrolase (RPE65), Sarcoglycan Alpha (SGCA), andsarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a). In some instances,the expression of a gene or expression or activity of a gene expressionproduct is inhibited by the administration of the composition to thesubject. In some instances, the expression of a gene or the expressionor the activity of a gene expression product is enhanced by theadministration of the composition to the subject.

In some cases, the composition is administered at dosage levelssufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, fromabout 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, fromabout 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg toabout 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject bodyweight per day, one or more times a day, to obtain the desiredtherapeutic effect.

In some cases, the viral genome (vg) concentration of the compositionthat is administered is between 1.0 ×10¹¹ vg per kilogram (kg) and 1.0×10¹⁶vg/kg. In some cases, the concentration of infectious particles ofat least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, or 10¹⁷. In some cases, the concentration of infectious particlesis 2×10⁷, 2×10⁸, 2×10⁹, 2×10¹⁰, 2×10¹¹, 2×10¹², 2×10¹³, 2×10¹⁴, 2×10¹⁵,2×10¹⁶, or 2×10¹⁷. In some cases, the concentration of the infectiousparticles 3×10⁷, 3×10⁸, 3×10⁹, 3×10¹⁰, 3×10¹¹, 3×10¹², 3×10¹³, 3×10¹⁴,3×10¹⁵, 3×10¹⁶, or 3×10¹⁷. In some cases, the concentration of theinfectious particles 4×10⁷, 4×10⁸, 4×10⁹, 4×10¹⁰, 4×10¹¹, 4×10¹²,4×10¹³, 4×10¹⁴, 4×10¹⁵, 4×10¹⁶, or 4×10¹⁷. In some cases, theconcentration of the infectious particles 5×10⁷, 5×10⁸, 5×10⁹, 5×10¹⁰,5×10¹¹, 5×10¹², 5×10¹³, 5×10¹⁴, 5×10¹⁵, 5×10¹⁶, or 5×10¹⁷. In somecases, the concentration of the infectious particles 6×10⁷, 6×10⁸,6×10⁹, 6×10¹⁰, 6×10¹¹, 6×10¹², 6×10¹³, 6×10¹⁴, 6×10¹⁵, 6×10¹⁶, or6×10¹⁷. In some cases, the concentration of the infectious particles7×10⁷, 7×10⁸, 7×10⁹, 7×10¹⁰, 7×10¹¹, 7×10¹², 7×10¹³, 7×10¹⁴, 7×10¹⁵,7×10¹⁶, or 7×10¹⁷. In some cases, the concentration of the infectiousparticles 8×10⁷, 8×10⁸, 8×10⁹, 8×10¹⁰, 8×10¹¹, 8×10¹², 8×10¹³, 8×10¹⁴,8×10¹⁵, 8×10¹⁶, or 8×10¹⁷. In some cases, the concentration of theinfectious particles 9×10⁷, 9×10⁸, 9×10⁹, 9×10¹⁰, 9×10¹¹, 9×10¹²,9×10¹³, 9×10¹⁴, 9×10¹⁵, 9×10¹⁶, or 9×10¹⁷.

In some embodiments, the administering of step is performed once.Alternatively, the administering of step is repeated at least twice. Theadministering of step may be performed once daily. In some cases, theadministering of step comprises intravenous administration. In somecases, the administering comprises pulmonary administration. In somecases, the administering comprises intranasal administration (such as aspray). In some cases, the administering of step comprises injecting thecomposition into a target in vivo environment. In some cases, theadministering of step does not comprise injecting the composition intothe target in vivo environment.

Subject

Disclosed herein methods of delivering at least one of an AAV particleand viral vector to a subject, for example—to treat or prevent a diseaseor condition in a subject. The subject, in some cases, is a mammal.Non-limiting examples of a mammal include a mouse, rat, guinea pig,rabbit, chimpanzee, or farm animal. In some instances, the mammal is anon-human primate. In some instances, the subject is human. The subjectof the present disclosure may not be diagnosed with a disease orcondition. Alternatively, the subject may be a patient that is diagnosedwith a disease or disorder, or suspected of having the disease or thedisorder.

Disease or Condition

Disclosed herein are methods of treating a disease or condition in asubject by administering a composition comprising a rAAV such as thosedisclosed herein. At least one advantage of the rAAVs disclosed herein,is that the rAAV may be used to treat virtually any disease or conditionthat would benefit from a transgene therapy, including but not limitedto spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS),Parkinson's disease, Pompe disease, Huntington's disease, Alzheimer'sdisease, Battens disease, lysosomal storage disorders, glioblastomamultiforme, Rett syndrome, Leber's congenital amaurosis, Late infantileneuronal ceroid lipofuscinosis (LINCL), chronic pain, stroke, spinalcord injury, traumatic brain injury and lysosomal storage disorders.

The disease or the condition may, in some embodiments, be characterizedby a reduced or ablated expression or activity of a gene or geneexpression product thereof, as compared to a normal individual. In someembodiments, be characterized by an increased or enhanced expression oractivity of a gene or gene expression product thereof, as compared to anormal individual.

In some cases, the disease or condition is localized to a particular invivo environment in the subject, e.g., the brain or the liver. Thecompositions of the present disclosure are particularly useful for thetreatment of the diseases or conditions described herein because theyspecifically target the in vivo environment and deliver a therapeuticnucleic acid engineered to modulate the activity or the expression of atarget gene expression product involved with the pathogenesis orpathology of the disease or condition.

In some instances, the disease or condition comprises a disease orcondition of the central nervous system (CNS). Non-limiting examples ofdisease of the CNS include Absence of the Septum Pellucidum, Acid LipaseDisease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, AcuteDisseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder(ADHD), Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis ofthe Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-GoutieresSyndrome Disorder, AIDS—Neurological Complications, Alexander Disease,Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease,Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, AngelmanSyndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia,Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation,Arteriovenous Malformation, Asperger Syndrome, Ataxia, AtaxiaTelangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration,Atrial Fibrillation and Stroke, Attention Deficit-HyperactivityDisorder, Autism Spectrum Disorder, Autonomic Dysfunction, Back Pain,Barth Syndrome, Batten Disease, Becker's Myotonia, Behcet's Disease,Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy,Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger'sDisease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus BirthInjuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brainand Spinal Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome,Bulbospinal Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathywith Subcortical Infarcts and Leukoencephalopathy (CADASIL), CanavanDisease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, CavernousAngioma, Cavernous Malformation, Central Cervical Cord Syndrome, CentralCord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis,Cephalic Disorders, Ceramidase Deficiency, Cerebellar Degeneration,Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis,Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavemous Malformation,Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy,Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-ToothDisease, Charcot-Marie-Tooth syndrome, classical rhizomelicchondrodysplasia punctata (RCDP), Chiari Malformation, Cholesterol EsterStorage Disease, Chorea, Choreoacanthocytosis, Chronic InflammatoryDemyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance,Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome,Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital FacialDiplegia, Congenital Myasthenia, Congenital Myopathy, CongenitalVascular Cavernous Malformations, Corticobasal Degeneration, CranialArteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt-JakobDisease, Cumulative Trauma Disorders, Cushing's Syndrome, CytomegalicInclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-DancingFeet Syndrome, Dandy-Walker Syndrome, Dawson Disease, Deafness, DeMorsier's Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia -Multi-Infarct, Dementia—Semantic, Dementia—Subcortical, Dementia With LewyBodies, Dentate Cerebellar Ataxia, Dentatorubral Atrophy,Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, DiabeticNeuropathy, Diffuse Sclerosis, Dravet Syndrome, Duchenne musculardystrophy, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia,Dyssynergia Cerebellaris Myoclonica, Dyssynergia CerebellarisProgressiva, Dystonias, Early Infantile Epileptic Encephalopathy, EmptySella Syndrome, Encephalitis, Encephalitis Lethargica, Encephaloceles,Encephalopathy, Encephalopathy (familial infantile), EncephalotrigeminalAngiomatosis, Epilepsy, Epileptic Hemiplegia, Erb's Palsy, Erb-Duchenneand Dejerine-Klumpke Palsies, Essential Tremor, ExtrapontineMyelinolysis, Fabry Disease, Fahr's Syndrome, Fainting, FamilialDysautonomia, Familial Hemangioma, Familial Idiopathic Basal GangliaCalcification, Familial Periodic Paralyses, Familial Spastic Paralysis,Farber's Disease, Febrile Seizures, Fibromuscular Dysplasia, FisherSyndrome, Floppy Infant Syndrome, Foot Drop, Friedreich's Ataxia,Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidoses,Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, GiantAxonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease,glioblastoma, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia,Glycogen Storage Disease, Guillain-Barre Syndrome, Hallervorden-SpatzDisease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm,Hemiplegia Alterans, Hereditary Neuropathies, Hereditary SpasticParaplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster,Herpes Zoster Oticus, Hirayama Syndrome, Holmes-Adie syndrome,Holoprosencephaly, HTLV-1 Associated Myelopathy, Hughes Syndrome,Huntington's Disease, Hydranencephaly, Hydrocephalus,Hydrocephalus—Normal Pressure, Hydromyelia, Hypercortisolism,Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-MediatedEncephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti,Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile PhytanicAcid Storage Disease, Infantile Refsum Disease, Infantile Spasms,Inflammatory Myopathies, Iniencephaly, Intestinal Lipodystrophy,Intracranial Cysts, Intracranial Hypertension, Isaacs' Syndrome, JoubertSyndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome,Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome(KTS), Kliiver-Bucy Syndrome, Korsakoff s Amnesic Syndrome, KrabbeDisease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton MyasthenicSyndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous NerveEntrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh'sDisease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy,Levine-Critchley Syndrome, Lewy Body Dementia, Lipid Storage Diseases,Lipoid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig'sDisease, Lupus—Neurological Sequelae, Lyme Disease—NeurologicalComplications, Machado- Joseph Disease, Macrencephaly, Megalencephaly,Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis,Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy,Microcephaly, Migraine, Miller Fisher Syndrome, Mini Stroke,Mitochondrial Myopathy, Moebius Syndrome, Monomelic Amyotrophy, MotorNeuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses,Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis,Multiple System Atrophy, Multiple System Atrophy with OrthostaticHypotension, Muscular Dystrophy, Myasthenia -Congenital, MyastheniaGravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy ofInfants, Myoclonus, Myopathy, Myopathy- Congenital, Myopathy-Thyrotoxic, Myotonia, Myotonia Congenita, Narcolepsy,Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation,Neurofibromatosis, Neuroleptic Malignant Syndrome, NeurologicalComplications of AIDS, Neurological Complications of Lyme Disease,Neurological Consequences of Cytomegalovirus Infection, NeurologicalManifestations of Pompe Disease, Neurological Sequelae Of Lupus,Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis,Neuronal Migration Disorders, Neuropathy- Hereditary, Neurosarcoidosis,Neurosyphilis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease,O′Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome,Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, OrthostaticHypotension, Overuse Syndrome, Pain -Chronic, PantothenateKinase-Associated Neurodegeneration, Paraneoplastic Syndromes,Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis, ParoxysmalHemicrania, Parry -Romberg, Pelizaeus-Merzbacher Disease, Pena ShokeirII Syndrome, Perineural Cysts, Periodic Paralyses, PeripheralNeuropathy, Periventricular Leukomalacia, Persistent Vegetative State,Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick'sDisease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors,Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome,Postherpetic Neuralgia, Postinfectious Encephalomyelitis, PosturalHypotension, Postural Orthostatic Tachycardia Syndrome, PosturalTachycardia Syndrome, Primary Dentatum Atrophy, Primary LateralSclerosis, Primary Progressive Aphasia, Prion Diseases, ProgressiveHemifacial Atrophy, Progressive Locomotor Ataxia, Progressive MultifocalLeukoencephalopathy, Progressive Sclerosing Poliodystrophy, ProgressiveSupranuclear Palsy, Prosopagnosia, Pseudo-Torch syndrome,Pseudotoxoplasmosis syndrome, Pseudotumor Cerebri, Psychogenic Movement,Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen'sEncephalitis, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease,Refsum Disease—Infantile, Repetitive Motion Disorders, Repetitive StressInjuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, RettSyndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome,Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease,Sandhoff Disease, Schilder's Disease, Schizencephaly, SeitelbergerDisease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia,Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome,Shingles, Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, SleepingSickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal CordInfarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal MuscularAtrophy, Spinocerebellar Atrophy, Spinocerebellar Degeneration,Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome,Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, SubacuteSclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy,Short-lasting, Unilateral, Neuralgiform (SUNCT) Headache, SwallowingDisorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis,Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, TabesDorsalis,Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, TemporalArteritis, Tethered Spinal Cord Syndrome, Thomsen's Myotonia, ThoracicOutlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis,Tourette Syndrome, Transient Ischemic Attack, Transmissible SpongiformEncephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor,Trigeminal Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome,Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis Syndromes of theCentral and Peripheral Nervous Systems, Von Economo's Disease, VonHippel-Lindau Disease (VHL), Von Recklinghausen's Disease, Wallenberg'sSyndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, WestSyndrome, Whiplash, Whipple's Disease, Williams Syndrome, WilsonDisease, Wolman's Disease, and X-Linked Spinal and Bulbar MuscularAtrophy.

In some instances, the disease or condition comprises a liver disease ordisorder, or is associated with a liver disease or disorder.Non-limiting examples include disorders of bile acid synthesis (e.g.,Wilson disease, Progressive familial intrahepatic cholestasis type 3),disorders of carbohydrate metabolism (e.g., Hereditary fructoseintolerance, Glycogen storage disease type IV), disorders of amino acidsmetabolism (e.g., tyrosinemia type I), Urea cycle disorders (e.g.,argininosuccinate lyase deficiency, citrin deficiency (CTLN2, NICCD)),disorders of lipid metabolism (e.g., cholesteryl ester storage disease),and others including but not limited to Alpha-1 antitrypsin deficiency,cystic fibrosis, hereditary hemochromatosis, Alström syndrome, andcongenital hepatic fibrosis.

In some instances, the disease or condition is a disease or condition isof the liver Non-limiting examples of liver diseases or disordersinclude Alagille Syndrome, Alcohol-Related Liver Disease, Alpha-1Antitrypsin Deficiency, Autoimmune Hepatitis, Benign Liver Tumors,Biliary Atresia, Cirrhosis, Crigler-Najjar Syndrome, Galactosemia,Gilbert Syndrome, Hemochromatosis, Hepatic Encephalopathy, Hepatitis A,Hepatitis B, Hepatitis C, Hepatorenal Syndrome, Intrahepatic Cholestasisof Pregnancy (ICP), Lysosomal Acid Lipase Deficiency (LAL-D), LiverCysts, Liver Cancer, Newborn Jaundice, Non-Alcoholic Fatty LiverDisease, Primary Biliary Cholangitis (PBC), Primary SclerosingCholangitis (PSC), Reye Syndrome, Type I Glycogen Storage Disease, andWilson Disease.

Provided here, are methods of treating a disease or a conditionassociated with an aberrant expression or activity of a target gene orgene expression product thereof, the method comprising modulating theexpression or the activity of a target gene or gene expression productin a subject by administering a rAAV encapsidating a heterologousnucleic acid of the present disclosure. In some instances,administration is systemic administration. In some instances, theexpression or the activity of the target gene or gene expression productis decreased, relative to that in a normal (non-diseased) individual;and administering the rAAV to the subject is sufficient to increase theexpression of the activity of the target gene or gene expression productto that of a normal individual. In some instances, the expression or theactivity of the gene or gene expression product is increased, relativeto that in a normal individual; and administering the rAAV to thesubject is sufficient to decrease the expression or the activity of thetarget gene or gene expression product. In a non-limiting example, asubject diagnosed with Alzheimer's disease, which is caused, in somecases, by a gain-of-function of a Presenilin 1 and/or Presenilin 2(encoded by the gene PSEN1 and PSEN2, respectively) is administered arAAV disclosed herein encapsidating a therapeutic nucleic acid that is asilencing RNA (siRNA), or other RNAi with a loss-of-function effect onPSEN1 mRNA.

Also provided are methods of treating or preventing a disease orcondition disclosed herein in a subject comprising administering to thesubject a therapeutically effective amount of an AAV vector comprising anucleic acid sequence encoding a therapeutic gene expression productdescribed herein. The AAV vector may be encapsidated in the modifiedcapsid protein or AAV viral particle described herein. In someinstances, the therapeutic gene expression product is effective tomodulate the activity or expression of a target gene or gene expressionproduct.

Formulations, Dosages, and Routes of Administration

In general, methods disclosed herein comprise administering atherapeutic rAAV composition by systemic administration. In someinstances, methods comprise administering a therapeutic rAAV compositionby oral administration. In some instances, methods compriseadministering a therapeutic rAAV composition by intraperitonealinjection. In some instances, methods comprise administering atherapeutic rAAV composition in the form of an anal suppository. In someinstances, methods comprise administering a therapeutic rAAV compositionby intravenous (“i.v.”) administration. It is conceivable that one mayalso administer therapeutic rAAV compositions disclosed herein by otherroutes, such as subcutaneous injection, intramuscular injection,intradermal injection, transdermal injection percutaneousadministration, intranasal administration, intralymphatic injection,rectal administration intragastric administration, intraocularadministration, intracerebro-ventricularl administration, intrathecally,or any other suitable parenteral administration. In some instances,methods comprise administering a therapeutic rAAV composition by topicaladministration, example, by brushing or otherwise contacting the rAAVcomposition to a region of the subject (e.g., eardrum, bladder). In someembodiments, routes for local delivery closer to site of injury orinflammation are preferred over systemic routes. Routes, dosage, timepoints, and duration of administrating therapeutics may be adjusted. Insome embodiments, administration of therapeutics is prior to, or after,onset of either, or both, acute and chronic symptoms of the disease orcondition.

An effective dose and dosage of pharmaceutical compositions to preventor treat the disease or condition disclosed herein is defined by anobserved beneficial response related to the disease or condition, orsymptom of the disease or condition. Beneficial response comprisespreventing, alleviating, arresting, or curing the disease or condition,or symptom of the disease or condition. In some embodiments, thebeneficial response may be measured by detecting a measurableimprovement in the presence, level, or activity, of biomarkers,transcriptomic risk profile, or intestinal microbiome in the subject. An“improvement,” as used herein refers to shift in the presence, level, oractivity towards a presence, level, or activity, observed in normalindividuals (e.g. individuals who do not suffer from the disease orcondition). In instances wherein the therapeutic rAAV composition is nottherapeutically effective or is not providing a sufficient alleviationof the disease or condition, or symptom of the disease or condition,then the dosage amount and/or route of administration may be changed, oran additional agent may be administered to the subject, along with thetherapeutic rAAV composition. In some embodiments, as a patient isstarted on a regimen of a therapeutic rAAV composition, the patient isalso weaned off (e.g., step-wise decrease in dose) a second treatmentregimen.

In some embodiments, pharmaceutical compositions in accordance with thepresent disclosure may be administered at dosage levels sufficient todeliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg,from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg toabout 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, fromabout 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weightper day, one or more times a day, to obtain the desired therapeutic,diagnostic, or prophylactic, effect. It will be understood that theabove dosing concentrations may be converted to vg or viral genomes perkg or into total viral genomes administered by one of skill in the art.

In some cases, a dose of the pharmaceutical composition may comprise aconcentration of infectious particles of at least or about 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, or 10¹⁷. In some cases,the concentration of infectious particles is 2×10⁷, 2×10⁸, 2×10⁹,2×10¹⁰, 2×10¹¹, 2×10¹², 2×10¹³, 2×10¹⁴, 2×10¹⁵, 2×10¹⁶, or 2×10¹⁷. Insome cases, the concentration of the infectious particles is 3×10⁷,3×10⁸, 3×10⁹, 3×10¹⁰, 3×10¹¹, 3×10¹², 3×10¹³, 3×10¹⁴, 3×10¹⁵, 3×10¹⁶, or3×10¹⁷. In some cases, the concentration of the infectious particles is4×10⁷, 4×10⁸, 4×10⁹, 4×10¹⁰, 4×10¹¹, 4×10¹², 4×10¹³, 4×10¹⁴, 4×10¹⁵,4×10¹⁶, or 4×10¹⁷. In some cases, the concentration of the infectiousparticles is 5×10⁷, 5×10⁸, 5×10⁹, 5×10¹⁰, 5×10¹¹, 5×10¹², 5×10¹³,5×10¹⁴, 5×10¹⁵, 5×10¹⁶, or 5×10¹⁷. In some cases, the concentration ofthe infectious particles is 6×10⁷, 6×10⁸, 6×10⁹, 6×10¹⁰, 6×10¹¹, 6×10¹²,6×10¹³, 6×10¹⁴, 6×10¹⁵, 6×10¹⁶, or 6×10¹⁷. In some cases, theconcentration of the infectious particles is 7×10⁷, 7×10⁸, 7×10⁹,7×10¹⁰, 7×10¹¹, 7×10¹², 7×10¹³, 7×10¹⁴, 7×10¹⁵, 7×10¹⁶, or 7×10¹⁷. Insome cases, the concentration of the infectious particles is 8×10⁷,8×10⁸, 8×10⁹, 8×10¹⁰, 8×10¹¹, 8×10¹², 8×10¹³, 8×10¹⁴, 8×10¹⁵, 8×10¹⁶, or8×10¹⁷. In some cases, the concentration of the infectious particles is9×10⁷, 9×10⁸, 9×10⁹, 9×10¹⁰, 9×10¹¹, 9×10¹², 9×10¹³, 9×10¹⁴, 9×10¹⁵,9×10¹⁶, or 9×10¹⁷.

Disclosed herein, in some embodiments are formulations ofpharmaceutically-acceptable excipients and carrier solutions suitablefor delivery of the rAAV compositions described herein, as well assuitable dosing and treatment regimens for using the particularcompositions described herein in a variety of treatment regimens. Insome embodiments, the amount of therapeutic gene expression product ineach therapeutically-useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable. In someinstances, the rAAV compositions are suitably formulated pharmaceuticalcompositions disclosed herein, to be delivered either intraocularly,intravitreally, parenterally, subcutaneously, intravenously,intracerebro-ventricularly, intramuscularly, intrathecally, orally,intraperitoneally, by oral or nasal inhalation, or by direct injectionto one or more cells, tissues, or organs by direct injection.

In some embodiments, the pharmaceutical forms of the AAV-based viralcompositions suitable for injectable use include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, and/orvegetable oils. Proper fluidity may be maintained, for example, by theuse of a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In some cases, for administration of an injectable aqueous solution, forexample, the solution may be suitably buffered, if necessary, and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity, andthe general safety and purity standards as required by FDA Office ofBiologics standards.

Disclosed herein are sterile injectable solutions comprising the rAAVcompositions disclosed herein, which are prepared by incorporating therAAV compositions disclosed herein in the required amount in theappropriate solvent with several of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. Injectable solutions may beadvantageous for systemic administration, for example by intravenousadministration.

Also provided herein are formulations in a neutral or salt form.Pharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

Pulmonary administration may be advantageously achieved via the buccaladministration. In some embodiments, formulations may comprise dryparticles comprising active ingredients. In such embodiments, dryparticles may have a diameter in the range from about 0.5 nm to about 7nm or from about 1 nm to about 6 nm. In some embodiments, formulationsmay be in the form of dry powders for administration using devicescomprising dry powder reservoirs to which streams of propellant may bedirected to disperse such powder. In some embodiments, self-propellingsolvent/powder dispensing containers may be used. In such embodiments,active ingredients may be dissolved and/or suspended in low-boilingpropellant in sealed containers. Such powders may comprise particleswherein at least 98% of the particles by weight have diameters greaterthan 0.5 nm and at least 95% of the particles by number have diametersless than 7 nm. Alternatively, at least 95% of the particles by weighthave a diameter greater than 1 nm and at least 90% of the particles bynumber have a diameter less than 6 nm. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form. Low boiling propellants generally includeliquid propellants having a boiling point of below 65 ° F. atatmospheric pressure. Generally, propellants may constitute 50% to 99.9%(w/w) of the composition, and active ingredient may constitute 0.1% to20% (w/w) of the composition. Propellants may further compriseadditional ingredients such as liquid non-ionic and/or solid anionicsurfactant and/or solid diluent (which may have particle sizes of thesame order as particles comprising active ingredients).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide active ingredients in the form of droplets of solution and/orsuspension. Such formulations may be prepared, packaged, and/or sold asaqueous and/or dilute alcoholic solutions and/or suspensions, optionallysterile, comprising active ingredients, and may conveniently beadministered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface-active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm. Formulations described hereinuseful for pulmonary delivery may also be useful for intranasaldelivery. In some embodiments, formulations for intranasaladministration comprise a coarse powder comprising the active ingredientand having an average particle size from about 0.2 μm to 500 μm. Suchformulations are administered in the manner in which snuff is taken,i.e. by rapid inhalation through the nasal passage from a container ofthe powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, comprise 0.1% to 20% (w/w) active ingredient, the balancecomprising an orally dissolvable and/or degradable composition and,optionally, one or more of the additional ingredients described herein.Alternately, formulations suitable for buccal administration maycomprise powders and/or an aerosolized and/or atomized solutions and/orsuspensions comprising active ingredients. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may comprise averageparticle and/or droplet sizes in the range of from about 0.1 nm to about200 nm, and may further comprise one or more of any additionalingredients described herein.

Suitable dose and dosage administrated to a subject is determined byfactors including, but not limited to, the particular therapeutic rAAVcomposition, disease condition and its severity, the identity (e.g.,weight, sex, age) of the subject in need of treatment, and can bedetermined according to the particular circumstances surrounding thecase, including, e.g., the specific agent being administered, the routeof administration, the condition being treated, and the subject or hostbeing treated.

The amount of AAV compositions and time of administration of suchcompositions will be within the purview of the skilled artisan havingbenefit of the present teachings. It is likely, however, that theadministration of therapeutically-effective amounts of the disclosedcompositions may be achieved by a single administration, example, asingle injection of sufficient numbers of infectious particles toprovide therapeutic benefit to the patient undergoing such treatment.This is made possible, at least in part, by the fact that certain targetcells (e.g., neurons) do not divide, obviating the need for multiple orchronic dosing.

Alternatively, in some circumstances, it may be desirable to providemultiple, or successive administrations of the AAV vector compositions,either over a relatively short, or a relatively prolonged period oftime, as may be determined by the medical practitioner overseeing theadministration of such compositions. For example, the number ofinfectious particles administered to a mammal may be on the order ofabout 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or even higher, infectiousparticles/ml given either as a single dose, or divided into two or moreadministrations as may be required to achieve therapy of the particulardisease or disorder being treated. In fact, in certain embodiments, itmay be desirable to administer two or more different AAV vectorcompositions, either alone, or in combination with one or more othertherapeutic drugs to achieve the desired effects of a particular therapyregimen. In various embodiments, the daily and unit dosages are altereddepending on a number of variables including, but not limited to, theactivity of the therapeutic rAAV composition used, the disease orcondition to be treated, the mode of administration, the requirements ofthe individual subject, the severity of the disease or condition beingtreated, and the judgment of the practitioner.

In some embodiments, the administration of the therapeutic rAAVcomposition is hourly, once every 2 hours, 3 hours, 4 hours, 5 hours, 6hours,? hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours,14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days,7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3years, 4 years, or 5 years, or 10 years. The effective dosage ranges maybe adjusted based on subject's response to the treatment. Some routes ofadministration will require higher concentrations of effective amount oftherapeutics than other routes.

Although not anticipated given the advantages of the present disclosure,in certain embodiments wherein the patient's condition does not improve,upon the doctor's discretion the administration of therapeutic rAAVcomposition is administered chronically, that is, for an extended periodof time, including throughout the duration of the patient's life inorder to ameliorate or otherwise control or limit the symptoms of thepatient's disease or condition. In certain embodiments wherein apatient's status does improve, the dose of therapeutic rAAV compositionbeing administered may be temporarily reduced or temporarily suspendedfor a certain length of time (i.e., a “drug holiday”). In specificembodiments, the length of the drug holiday is between 2 days and 1year, including by way of example only, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or morethan 28 days. The dose reduction during a drug holiday is, by way ofexample only, by 10%-100%, including by way of example only 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, and 100%. In certain embodiments, the dose of drug beingadministered may be temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug diversion”). In specificembodiments, the length of the drug diversion is between 2 days and 1year, including by way of example only, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or morethan 28 days. The dose reduction during a drug diversion is, by way ofexample only, by 10%-100%, including by way of example only 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, and 100%. After a suitable length of time, the normal dosingschedule is optionally reinstated.

In some embodiments, once improvement of the patient's conditions hasoccurred, a maintenance dose is administered if necessary. Subsequently,in specific embodiments, the dosage or the frequency of administration,or both, is reduced, as a function of the symptoms, to a level at whichthe improved disease, disorder or condition is retained. In certainembodiments, however, the patient requires intermittent treatment on along-term basis upon any recurrence of symptoms.

Toxicity and therapeutic efficacy of such therapeutic regimens aredetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD50 and the EDS°. The dose ratio between the toxic andtherapeutic effects is the therapeutic index and it is expressed as theratio between LD50 and EDS°. In certain embodiments, the data obtainedfrom cell culture assays and animal studies are used in formulating thetherapeutically effective daily dosage range and/or the therapeuticallyeffective unit dosage amount for use in mammals, including humans. Insome embodiments, the dosage amount of the therapeutic rAAV compositiondescribed herein lies within a range of circulating concentrations thatinclude the ED50 with minimal toxicity. In certain embodiments, thedaily dosage range and/or the unit dosage amount varies within thisrange depending upon the dosage form employed and the route ofadministration utilized.

Additional Therapeutic

A therapeutic nucleic acid may be used alone or in combination with anadditional therapeutic agent (together, “therapeutic agents”). In somecases, an “additional therapeutic agent” as used herein is administeredalone. The therapeutic agent may be administered together orsequentially in a combination therapy. The combination therapy may beadministered within the same day, or may be administered one or moredays, weeks, months, or years apart. In some cases, a therapeuticnucleic acid provided herein is administered if the subject isdetermined to be non-responsive to a first line of therapy.

The additional therapeutic agent can comprise a small molecule. Theadditional therapeutic agent can comprise an antibody, orantigen-binding fragment. The additional therapeutic agent can comprisea cell-based therapy. Exemplary cell-based therapies include withoutlimitation immune effector cell therapy, chimeric antigen receptorT-cell (CAR-T) therapy, natural killer cell therapy and chimeric antigenreceptor natural killer (NK) cell therapy. Either NK cells, or CAR-NKcells, or a combination of both NK cells and CAR-NK cells can be used incombination with the methods disclosed herein. In some embodiments, theNK cells and CAR-NK cells are derived from human induced pluripotentstem cells (iPSC), umbilical cord blood, or a cell line. The NK cellsand CAR-NK cells can comprise a cytokine receptor and a suicide gene.The cell-based therapy can comprises a stem cell therapy. The stem celltherapy may be embryonic or somatic stem cells. The stem cells may beisolated from a donor (allogeneic) or isolated from the subject(autologous). The stem cells may be expanded adipose-derived stem cells(eASCs), hematopoietic stem cells (HSCs), mesenchymal stem (stromal)cells (MSCs), or induced pluripotent stem cells (iPSCs) derived from thecells of the subject.

III. KITS

Disclosed herein are kits comprising compositions disclosed herein. Alsodisclosed herein are kits for the treatment or prevention of a diseaseor conditions of the central nervous system (CNS), or target organ orenvironment (e.g., liver). In some instances, the disease or conditionis cancer, a pathogen infection, pulmonary disease or condition,neurological disease, muscular disease, or an immune disorder, such asthose described herein. In one embodiment, a kit can include atherapeutic or prophylactic composition containing an effective amountof a composition of a rAAV particle encapsidating a recombinant AAVvector encoding a therapeutic nucleic acid (e.g., therapeutic nucleicacid) and a recombinant AAV (rAAV) capsid protein of the presentdisclosure. In another embodiment, a kit can include a therapeutic orprophylactic composition containing an effective amount of cellsmodified by the rAAV described herein (“modified cell”), in unit dosageform that express therapeutic nucleic acid. In some embodiments, a kitcomprises a sterile container which can contain a therapeuticcomposition; such containers can be boxes, ampules, bottles, vials,tubes, bags, pouches, blister-packs, or other suitable container formsknown in the art. Such containers can be made of plastic, glass,laminated paper, metal foil, or other materials suitable for holdingmedicaments.

In some cases, rAAV are provided together with instructions foradministering the rAAV to a subject having or at risk of developing thedisease or condition (e.g., disease of the CNS, PNS, liver, and thelike). Instructions can generally include information about the use ofthe composition for the treatment or prevention of the disease orcondition.

In some cases, a kit can include allogenic cells. In some cases, a kitcan include cells that can comprise a genomic modification. In somecases, a kit can comprise “off-the-shelf” cells. In some cases, a kitcan include cells that can be expanded for clinical use. In some cases,a kit can contain contents for a research purpose.

In some cases, the instructions include at least one of the following:description of the therapeutic rAAV composition; dosage schedule andadministration for treatment or prevention of the disease or conditiondisclosed herein; precautions; warnings; indications;counter-indications; overdosage information; adverse reactions; animalpharmacology; clinical studies; and/or references. The instructions canbe printed directly on the container (when present), or as a labelapplied to the container, or as a separate sheet, pamphlet, card, orfolder supplied in or with the container. In some cases, instructionsprovide procedures for administering the rAAV to the subject alone. Insome cases, instructions provide procedures for administering the rAAVto the subject at least about 1 hour (hr), 2 hr, 3 hr, 4 hr, 5 hr, 6 hr,7 hr, 8 hr, 9 hr,10 hr, 11 hr, 12 hr, 13 hr, 14 hr, 15 hr, 16 hr, 17 hr,18 hr, 19 hr, 20 hr, 21 hr, 22 hr, 23 hr, 24 hr, 25 hr, 26 hr, 27 hr, 28hr, 29 hr, 30 hr, or up to 2 days, 3 days, 4 days, 5 days, 6 days, or 7days after or before administering an additional therapeutic agentdisclosed herein. In some instances, the instructions provide that therAAV is formulated for intravenous injection.

IV. DEFINITIONS

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description and/or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the given value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” should be assumed to mean an acceptable error range for theparticular value.

As used herein “consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the stated purpose. Thus,a composition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed disclosure, such ascompositions for treating skin disorders like acne, eczema, psoriasis,and rosacea.

The terms “homologous,” “homology,” or “percent homology” are usedherein to generally mean an amino acid sequence or a nucleic acidsequence having the same, or similar sequence to a reference sequence.Percent homology of sequences can be determined using the most recentversion of BLAST, as of the filing date of this application.

The terms “increased,” or “increase” are used herein to generally meanan increase by a statically significant amount. In some embodiments, theterms “increased,” or “increase,” mean an increase of at least 10% ascompared to a reference level, for example an increase of at least about10%, at least about 20%, or at least about 30%, or at least about 40%,or at least about 50%, or at least about 60%, or at least about 70%, orat least about 80%, or at least about 90% or up to and including a 100%increase or any increase between 10-100% as compared to a referencelevel, standard, or control. Other examples of “increase” include anincrease of at least 2-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or moreas compared to a reference level.

The terms, “decreased” or “decrease” are used herein generally to mean adecrease by a statistically significant amount. In some embodiments,“decreased” or “decrease” means a reduction by at least 10% as comparedto a reference level, for example a decrease by at least about 20%, orat least about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% decrease (e.g., absentlevel or non-detectable level as compared to a reference level), or anydecrease between 10-100% as compared to a reference level. In thecontext of a marker or symptom, by these terms is meant a statisticallysignificant decrease in such level. The decrease can be, for example, atleast 10%, at least 20%, at least 30%, at least 40% or more, and ispreferably down to a level accepted as within the range of normal for anindividual without a given disease.

The terms “subject” is any organism. In some instances, the organism isa mammal. Non-limiting examples of mammal include, any member of themammalian class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. In one aspect, the mammal is a human. The term“animal” as used herein comprises human beings and non-human animals. Inone embodiment, a “non-human animal” is a mammal, for example a rodentsuch as rat or a mouse. In some instances, the subject is a patient,which as used herein, may refer to a subject diagnosed with a particulardisease or disorder.

The term “gene,” as used herein, refers to a segment of nucleic acidthat encodes an individual protein or RNA (also referred to as a “codingsequence” or “coding region”), optionally together with associatedregulatory region such as promoter, operator, terminator and the like,which may be located upstream or downstream of the coding sequence.

The term “adeno-associated virus,” or “AAV” as used herein refers to theadeno-associated virus or derivatives thereof. Non-limited examples ofAAV's include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3),AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7(AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAVtype 11 (AAV11), AAV type 12 (AAV12), avian AAV, bovine AAV, canine AAV,equine AAV, primate AAV, non-primate AAV, and ovine AAV. In someinstances, the AAV is described as a “Primate AAV,” which refers to AAVthat infect primates. Likewise an AAV may infect bovine animals (e.g.,“bovine AAV”, and the like). In some instances, the AAV is wildtype, ornaturally occurring. In some instances, the AAV is recombinant.

The term “AAV capsid” as used herein refers to a capsid protein orpeptide of an adeno-associated virus. In some instances, the AAV capsidprotein is configured to encapsidate genetic information (e.g., atransgene, therapeutic nucleic acid, viral genome). In some instances,the AAV capsid of the instant disclosure is a modified AAV capsid,relative to a corresponding parental AAV capsid protein.

The term “tropism” as used herein refers to a quality or characteristicof the AAV capsid that may include specificity for, and/or an increaseor a decrease in efficiency of, expressing the encapsidated geneticinformation into one in in vivo environment, relative to a second invivo environment. An in vivo environment, in some instances, is acell-type. An in vivo environment, in some instances, is an organ ororgan system.

The term “AAV vector” as used herein refers to nucleic acid polymerencoding genetic information related to the virus. The AAV vector may bea recombinant AAV vector (rAAV), which refers to an AAV vector generatedusing recombinatorial genetics methods. In some instances, the rAAVvector comprises at least one heterologous polynucleotide (e.g. apolynucleotide other than a wild-type or naturally occurring AAV genomesuch as a transgene).

The term “AAV particle” as used herein refers to an AAV virus, virion,AAV capsid protein or component thereof. In some cases, the AAV particleis modified relative to a parental AAV particle.

The term “gene product” of “gene expression product” refers to anexpression product of a polynucleotide sequence such as, for e.g., apolypeptide, peptide, protein or RNA, including interfering RNA (e.g.,siRNA, miRNA, shRNA) and messenger RNA (mRNA).

The terms “operatively linked” or “operably linked” refers to a locationof two or more elements being close together, and in some cases, next toone other (e.g., genetic elements such as a promoter, enhancer,termination signal sequence, polyadenylation sequence, and the like)that enables a functional relationship between the two or more elements.In one non-limiting example, a promoter that is operatively linked to acoding region enables the initiation of transcription of the codingsequence.

The term “heterologous” as used herein refers to a genetic element(e.g., coding region) or gene expression product (e.g., RNA, protein)that is derived from a genotypically distinct entity from that of therest of the entity to which it is being compared.

The term “endogenous” as used herein refers to a genetic element (e.g.,coding region) or gene expression product (e.g., RNA, protein) that isnaturally occurring in or associated with an organism or a particularcell within the organism.

A “detectable moiety” as used herein refers to a moiety that can becovalently or noncovalently attached to a compound or biomolecule thatcan be detected for instance, using techniques known in the art. Inembodiments, the detectable moiety is covalently attached. Thedetectable moiety may provide for imaging of the attached compound orbiomolecule. The detectable moiety may indicate the contacting betweentwo compounds. Exemplary detectable moieties are fluorophores,antibodies, reactive dies, radio-labeled moieties, magnetic contrastagents, and quantum dots. Exemplary fluorophores include fluorescein,rhodamine, GFP, coumarin, FITC, Alexa fluor, Cy3, Cy5, BODIPY, andcyanine dyes. Exemplary radionuclides include Fluorine-18, Gallium-68,and Copper-64. Exemplary magnetic contrast agents include gadolinium,iron oxide and iron platinum, and manganese.

The terms “treat,” “treating,” and “treatment” as used herein refers toalleviating or abrogating a disorder, disease, or condition; or one ormore of the symptoms associated with the disorder, disease, orcondition; or alleviating or eradicating a cause of the disorder,disease, or condition itself. Desirable effects of treatment caninclude, but are not limited to, preventing occurrence or recurrence ofdisease, alleviation of symptoms, diminishing any direct or indirectpathological consequences of the disease, preventing metastasis,decreasing the rate of disease progression, amelioration or palliationof the disease state and remission or improved prognosis.

The term “therapeutically effective amount” refers to the amount of acompound or therapy that, when administered, is sufficient to preventdevelopment of, or alleviate to some extent, one or more of the symptomsof a disorder, disease, or condition of the disease; or the amount of acompound that is sufficient to elicit biological or medical response ofa cell, tissue, system, animal, or human that is being sought by aresearcher, veterinarian, medical doctor, or clinician.

The term “pharmaceutically acceptable carrier,” “pharmaceuticallyacceptable excipient,” “physiologically acceptable carrier,” or“physiologically acceptable excipient” refers to a pharmaceuticallyacceptable material, composition, or vehicle, such as a liquid or solidfiller, diluent, excipient, solvent, or encapsulating material. Acomponent can be “pharmaceutically acceptable” in the sense of beingcompatible with the other ingredients of a pharmaceutical formulation.It can also be suitable for use in contact with the tissue or organ ofhumans and animals without excessive toxicity, irritation, allergicresponse, immunogenicity, or other problems or complications,commensurate with a reasonable benefit/risk ratio. See, Remington: TheScience and Practice of Pharmacy, 21st Edition; Lippincott Williams &Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients,5th Edition; Rowe et al., Eds., The Pharmaceutical Press and theAmerican Pharmaceutical Association: 2005; and Handbook ofPharmaceutical Additives, 3rd Edition; Ash and Ash Eds., GowerPublishing Company: 2007; Pharmaceutical Preformulation and Formulation,Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004).

The term “pharmaceutical composition” refers to a mixture of a compounddisclosed herein with other chemical components, such as diluents orcarriers. The pharmaceutical composition can facilitate administrationof the compound to an organism. Multiple techniques of administering acompound exist in the art including, but not limited to, oral,injection, aerosol, parenteral, and topical administration.

Non-limiting examples of “sample” include any material from whichnucleic acids and/or proteins can be obtained. As non-limiting examples,this includes whole blood, peripheral blood, plasma, serum, saliva,mucus, urine, semen, lymph, fecal extract, cheek swab, cells or otherbodily fluid or tissue, including but not limited to tissue obtainedthrough surgical biopsy or surgical resection. In various embodiments,the sample comprises tissue from the large and/or small intestine. Invarious embodiments, the large intestine sample comprises the cecum,colon (the ascending colon, the transverse colon, the descending colon,and the sigmoid colon), rectum and/or the anal canal. In someembodiments, the small intestine sample comprises the duodenum, jejunum,and/or the ileum. Alternatively, a sample can be obtained throughprimary patient derived cell lines, or archived patient samples in theform of preserved samples, or fresh frozen samples.

The term “in vivo” is used to describe an event that takes place in asubject's body.

The term “ex vivo” is used to describe an event that takes place outsideof a subject's body. An ex vivo assay is not performed on a subject.Rather, it is performed upon a sample separate from a subject. Anexample of an ex vivo assay performed on a sample is an “in vitro”assay.

The term “in vitro” is used to describe an event that takes placescontained in a container for holding laboratory reagent such that it isseparated from the biological source from which the material isobtained. In vitro assays can encompass cell-based assays in whichliving or dead cells are employed. In vitro assays can also encompass acell-free assay in which no intact cells are employed.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

V. EXAMPLES Example 1 Method of Producing an rAAV

A recombinant AAV (rAAV) is produced. Three plasmid vectors aretriple-transfected into an immortalized HEK293 using a standardtransfection protocol (e.g., with PEI). The first vector contains atransgene cassette flanked by inverted terminal repeat (ITR) sequencesfrom a parental AAV virus. The transgene cassette has a promotersequence and that drives transcription of a heterologous nucleic acid inthe nucleus of the target cell. The second vector contains nucleic acidsencoding the AAV Rep gene, as well as a modified Cap gene e.g., AAV2/9REP-AAP-ACap). Modified Cap gene comprises any one of the DNA sequencesprovided in FIGS. 33-35, which are the DNA sequences encoding themodified AAV capsid proteins of the present disclosure. The third vectorcontains nucleic acids encoding helper virus proteins needed for viralassembly, and packaging of the heterologous nucleic acid into themodified capsid structure.

Viral particles are harvested from the media after 72 h posttransfection and from the cells and media at 120 h post transfection.Virus present in the media is concentrated by precipitation with 8%poly(ethylene glycol) and 500 mM sodium chloride and the precipitatedvirus is added to the lysates prepared from the collected cells. Theviruses are purified over iodixanol (Optiprep, Sigma) step gradients(15%, 25%, 40% and 60%). Viruses are concentrated and formulated in PBS.Virus titers are determined by measuring the number of DNasel-resistantvector genome copies (VGs) using qPCR and the linearized genome plasmidas a control.

Example 2 Methods of Identifying Variant AAV Capsid Proteins

Plasmid

Library generation. The rAAV-ACap-in-cis-Lox2 plasmid (FIG. 36) is amodification of the rAAV-ACap-in-cis-Lox plasmid. For 7-mer-i libraryfragment generation, the pCRII-9Cap-XE plasmid was used as a template.The AAV2/9 REP-AAP-ACap plasmid (FIG. 36) was modified from the AAV2/9REP-AAP plasmid.

The rAAV-ACap-in-cis-Lox2 plasmid consists of three major elements thatare flanked by AAV2 ITRs. (i) UBC ubiquitous promoter driving theexpression of fluorescent protein, mNeongreen, followed by a syntheticpolyadenylation sequence. The mCherry expression cassette of theprevious version of the plasmid was replaced by mNeonGreen cassette.(ii) A portion of AAV2 rep gene that has the splicing sequences and AAVSp41 promoter (1680-1974 residues of GenBank AF085716.1) followed by AAV9cap gene. The prior version of this plasmid, rAAV-ACap-in-cis-Lox, has ashort 12 bp sequence between restriction sites Xbal and Agel at AA 450and 592 of the AAV9 Cap gene. This was replaced by a 723 bp sequence ofmRuby2 gene in-frame (acts as filler DNA) in the newer version of theplasmid. (iii) SV40 polyadenylation sequence that is flanked by lox71and lox66 sites. The minor changes were introduced to the prior versionof the plasmid to facilitate ease of cloning and to visualize mammaliancell transfection. The Lox sites in these rAAV plasmids show modestlevels of Cre-independent flipping. This was minimized during PCR-basedcapsid recovery by lowering the number of amplification cycles to apoint where we cannot recover any rAAV capsids from the control DNAextracted from wild-type mice (i.e., lacking Cre expression) that wereinjected with the library. The pCRII-9Cap-XE plasmid contains the AAV9capsid gene sequence from AAs 450-592 and is flanked by Xbal and Agelrestriction sites.

The AAV2/9 REP-AAP-ACap plasmid has the five previously existing stopcodons of AAV2/9 REP-AAP in addition to the deletion of AAs 450-592 ofthe AAV9 capsid sequence. These modifications did not affect vectorproduction. The deletion of the overlapping fragment between the REP-AAPand rAAV-ACap-in-cis-Lox2 plasmids minimizes recombination betweenplasmids that could potentially generate AAV9 wild-type capsids duringco-transfection in vector production.

Capsid Characterization

AAV capsids. The AAV capsid variants with 7-mer insertions or 11-mersubstitutions were made between positions 587-597 of AAV-PHP.B capsidusing the pUCmini-iCAP-PHP.B backbone (Addgene ID: 103002).

ssAAV genomes. To characterize the AAV capsid variants, the singlestranded (ss) rAAV genomes were used. Genomes such aspAAV:CAG-mNeonGreen27 (equivalent plasmid, pAAV: CAG-eYFP35; Addgene ID:104055), pAAV:CAG-NLS-EGFP26 (equivalent version with one NLS is onAddgene ID 104061), pAAV:CAG-DIO-EYFP35 (Addgene ID: 104052), pAAV:GfABC1D-2xNLS-mTurquoise235 (Addgene ID: 104053), and pAAV-Ple261-iCre30(Addgene ID 49113) were used.

pAAV:CAG-mNeonGreen2 genome consists of a ubiquitousCMV-(3-Actin-intron-(3-Globin (CAG) hybrid promoter driving theexpression of a fluorescent protein, mNeonGreen (equivalent plasmid,pAAV: CAG-eYFP3; Addgene ID: 104055). pAAV:CAG-NLS-EGFP1 consists of NLSsequences at the N- and C-termini of EGFP and is driven by the CAGpromoter. An equivalent version with one NLS is on Addgene (ID 104061).pAAV:CAG-DIO-EYFP3 (Addgene ID: 104052) consists of a EYFP gene built inthe reverse direction of the CAG promoter, and it is flanked by a pairof Cre-Lox sites (Lox P and Lox 2272) on either ends.

In cells expressing Cre, the Cre-lox pair inverts EYFP enablingtranscription and translation, followed by excision in the lox site toprevent re-inversion. pAAV: GfABC1D-2xNLS-mTurquoise23, referred toelsewhere as pAAV:GFAP-2xNLS-mTurquoise2 (Addgene ID: 104053), consistsof NLS sequences at the N- and C-termini of mTurquoise2 and is driven bythe astrocyte-specific promoter GfABC1D4. pAAV:Ple261-iCre5 (Addgene ID49113) contains an endothelial-cell-specific promoter driving theexpression of iCre.

pAAV:CAG-XFP (mNeongreen) was packaged for characterizing AAV variants.However, when performing quantification of cell-types: neurons,astrocytes and oligodendrocytes, CAG-NLS-EGFP was used to restrict theexpression to nucleus for easier quantification using microscope images.GFAP-NLS-mTurq2 is used to quantify astrocytes. CAG-DIO-EYFP is used forCre driver lines, due to the presence of lox sites in this plasmid.

The self-complementary genome from Dr. Guangping Gao, scAAV:CB6-EGFPgenome has a hybrid ubiquitous CB6 promoter (975 bp) comprising a CMVenhancer (cytomegalovirus immediate early enhancer), a chicken-f3-actinpromoter and hybrid intron, that drives the expression of EGFP. Thegenome has a rabbit globin poly A (127 bp) following the EGFP gene. ThescAAV:CAG-EGFP (Addgene ID:83279), vector uses a ubiquitousCMV-β-Actin-intron-β-Globin (CAG) hybrid promoter to drive theexpression of EGFP.

Round-I AAV Capsid Library Generation

Mutagenesis strategy. The 7-mer randomized insertion was designed usingthe NNK saturation mutagenesis strategy, involving degenerate primers(from Integrated DNA Technologies, Inc.) containing mixed bases. N canbe A, C, G, or T bases and K can be G, or T. Using this strategy,combinations of all 20 AAs at each position of the 7-mer peptide using33 codons were obtained, resulting in a library size of 1.28 billion atthe level of AA combinations. The mutagenesis strategy for the 3-mer-sPHP.B library is described in Chan, K. Y. et al. Engineered AAVs forefficient noninvasive gene delivery to the central and peripheralnervous systems. Nat. Neurosci. 20, 1172-1179 (2017).

Library cloning. The 480 bp AAV capsid fragment (450-592 AAs) with the7-mer random insertion between AAs 588 and 589 was generated byconventional PCR methods with a mixed base degenerate primer. Thelibrary fragment was amplified from the pCRII-9Cap-XE template by Q5 HotStart High-Fidelity 2× Master Mix (NEB; M0494S) with forward primer, XF:5′-ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC-3′ and reverse primer,7×MNN-588i: 5′-GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNNMNNMNNMNNMNNTTGGGCACTCTGGTGGTTTGTG-3′. To avoid PCR-induced biases resultingfrom point mutations, recombination, and template switching, PCRamplification was limited to 15-20 cycles and the reactions were scaledup to get the required yield. The resulting PCR products were run on a1% agarose gel and extracted with a Zymoclean Gel DNA Recovery kit (ZymoResearch; D4007). It is critical to avoid AAV contamination during thisstep by taking precautionary measures like using a clean gel-running boxand freshly prepared 1×TAE buffer.

rAAV-ACap-in-cis-Lox2 plasmid (6960 bp) was linearized with therestriction enzymes Agel and Xbal by following the NEB recommendedprotocol for double digestion. The digested plasmid was run on a 0.8%-1%agarose gel to extract the linearized backbone (6237 bp) with aZymoclean Gel DNA Recovery kit.

The amplified library fragment was assembled into the linearized vectorwith the NEBuilder HiFi DNA Assembly Master Mix (NEB; E2621S) and a 1:2molar ratio of vector to insert, to assemble at 50° C. for 60 min.

Library purification. The assembled library was then subjected toPlasmid Safe (PS) DNase (Epicentre; E3105K) treatment to purify theassembled product by degrading the un-assembled DNA fragments from themixture. For the R1 library, around 3U of PS DNase per 1 μg of input DNAwas used in a 30 min reaction at 37° C. Alternatively, Exonuclease V(RecBCD) was used following the NEB recommended protocol (NEB; M0345S).Both procedures yielded comparable results. The resulting mixture wasfurther purified with a DNA Clean and Concentrator kit (Zymo Research;D4013).

Library yield. With an assembly efficiency of 15%-20% post-PS treatment,a yield of about 15-20 ng per 100 ng of input DNA per 20 μL reaction wasobtained. For building the 7-mer-i DNA library, approximately 5-6 μg ofinput DNA was used to obtain around 800 ng of assembled library.

Quality control. To validate successful assembly of the library, 1 ng ofthe final assembled library was transformed into E. coli SURE 2Supercompetent Cells (Integrated Sciences; 200152). Colonies on anLB/Agar plate containing carbenicillin antibiotic after overnightincubation at 37° C. were identified. The DNA library was sequencedaround the insertion site (Laragen; Sanger Sequencing). A non-biasedlibrary shows multiple nucleotide peaks of equal diversity (25% each ofA, T, G, C) across every base position of the diversified region. Toverify that the ITRs were intact, Smal digestion was carried out as perthe NEB recommended protocol (NEB; R0141S). To validate successfultransfection and assess the vector-production yield per 150 mm dish, 10ng of 7-mer-i library was used to transfect HEK293 producer cells.Uniform expression of mNeonGreen protein across HEK cells was observed,and an average yield of 0.1-1×10¹¹ vg was obtained per 150 mm dish.Using the average yield per dish, the vector production for in vivoselection was scaled up (See FIG. 45).

Round-2 AAV Capsid DNA Library

PCR pool design. To maintain proportionate pooling, the fraction of eachsample/library that needs to be pooled based on an individual library'sdiversity was mathematically determined. This process involvedestimation of the diversity precluding noise and consideration ofamplification of this diversity across samples by determining the areaunder the curve for the interval of high-confidence variants that fallsin the higher RC range. The area under the curve (AUC) was estimatedusing the composite Simpson's rule by plotting all the recoveredvariants in a library (X-coordinate) to their read counts (RCs or copynumber from deep sequencing data, Y-coordinate) (see FIG. 40). Todetermine the definite intervals for AUC, the data was sorted based onthe decreasing order of the RCs. Noticeably, the distribution has twophases, with a steadier slope of variants in the higher RC range,followed by a steep drop in the slope of the curve (˜50-1000 fold lowerRCs). By observation, this steeper side of the curve is predominant insequencing errors/ PCR mutations, hence this error dominant slopeotherwise called noise from was precluded from the AUC estimation. Whencomparing composite Simpson's rule with another function, such ascomposite trapezoidal rule, the difference was miniscule.

This area is then used to determine the fraction of an individuallibrary that needs to be pooled into PCR pool library using the formula:[Area under the curve/ total number of libraries pooled].

The pooled sample was used as a template for further amplification with12 cycles of 98° C. for 10 s, 60° C. for 20 s, and 72° C. for 30 s by Q5polymerase, using the primers 588-R2lib-F:5′-CACTCATCGACCAATACTTGTACTATCTCTCT-3′ and 588-R2lib-R:5′-GTATTCCTTGGTTTTGAACCCAACCG-3′. Similar to R1 library generation, thePCR product was assembled into the rAAV-ACap-in-cis-Lox2 plasmid and thevirus was produced.

The R1 libraries used to build R2 were the Cre-Lox flipped rAAV DNA fromhalf of the mouse brains (˜0.3 g) and portion of spinal cords (0.1-0.2g) from all Cre lines. The amount of tissue processed here wassufficient for complete capsid library recovery. The differentiallypooled and amplified libraries (by PCR pool or synthetic pool) wereassembled using gibson assembly with a follow-up PS or Exonuclease Vtreatment (as described in R1 library generation). Successful librarygeneration was validated by transformation, Sanger sequencing, and anITR Smal digest.

For vector production, about 10 ng of the purified and assembled librarywas used to transfect each 150 mm dish of 293T cells, and a yield ofabout 6×10¹¹ vg per 150 mm dish was obtained (i.e. the R2 yield was sixtimes that of R1, unsurprisingly given these sequences have alreadyproduced well enough to survive R1 selection).

Synthetic pool design. As described in the PCR pool strategy,high-confidence variants were chosen with RCs above the error-dominantnoise slope from the plot of library distribution (see FIG. 40). Thiscame to about 9000 sequences from all brain and spinal cord samples ofall Cre lines. A similar primer design as mentioned in the descriptionof the R1 library generation was used. Primers XF:5′-ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC-3′ and 11-mer-588i:5′-GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCXXXXXXMNNMNNMNNMNNMNNMNNMNNXXXXXXACTCTGGTGGTTTGTG-3′, where“XXXXXXMNNMNNMNNMNNMNNMNNMNNXXXXXX” was replaced with unique nucleotidesequence of a 7-mer tissue recovered variant (7×MNN) along withmodification of two adjacent codons flanking on either end of the 7-merinsertion site (6xX), which are residues 587-588 “AQ” and residues589-590 “AQ” on AAV9 capsid. To select sequences for synthesis, the R1brain and spinal cord libraries were chosen and used the same thresholdlimit for RC as described in the PCR pool strategy. This came to about9000 sequences from all brain and spinal cord samples of all Cre lines.Since spike-in library has 11-mer mutated variants, the same primerdesign where “XXXXXXMNNMNNMNNMNNMNNMNNMNNXXXXXX” was replaced with aspecific nucleotide sequence of a 11-mer variant. A duplicate of eachsequence in this library was designed with different codons optimizedfor mammals. The primers were designed using a custom built python basedscript (code will be made available in Github). The custom-designedoligopool was synthesized in an equimolar ratio by Twist Biosciences.The oligopool was used to amplify the pCRII-XE Cap9 template over 13cycles of 98° C. for 10 s, 60° C. for 20 s, and 72° C. for 30 s. Toobtain a higher yield for large-scale library preparation, the productof the first PCR was used as a template for the second PCR using theprimers XF and 588-R2lib-R (described above) and amplified for 13cycles. As described in the description of the R1 library generation,the PCR product was assembled into an rAAV backbone and processed andpurified for virus production. About 10 ng per 150 mm dish of HEK293cells produced about 6×10¹¹ vg of virus library.

AAV Viral Library Production and Purification

To prevent capsid mosaic formation of the 7-mer-i library in HEK293producer cells, only 10 ng of assembled library per 150 mm dish weretransfected along with other required reagents for AAV vectorproduction. In addition to the 10 ng of library transfection per 150 mmdish of 293T producer cells, three plasmids were transfected: AAV2/9REP-AAP-ACap, pUC18 and pHelper (genes encoding adenoviral proteins forAAV replication) at a ratio of 1:1:2. The plasmid pUC18 acts as a fillerDNA to compensate for the low amount of library DNA in order to maintainthe N:P ratio required for optimal transfection using polyethylenimine(PEI, Polysciences; 24765-1) transfection). The cells and culture mediawere harvested at 60 h post-transfection to collect the viral particles.rAAV harvest and purification were performed as per the protocol. Thesmall amount of library DNA per plate and early cell harvest time arecritical for reducing the possibility of mosaic capsid assemblies duringvector production (similar considerations seen in prior reports).

For 7-mer-i library, the production was scaled up to 60 dishes (˜1.8×10⁷cells/dish) and with ˜10% transfected with the library, resulted in˜1×10⁸ total transformants. For an NNK 7mer library with ˜1×10⁸ totaltransformants, the number of unique variants is 9.99×10⁷.

For the rAAV DNA extraction from purified rAAV viral library, ˜10% ofthe purified viral library was used to extract the viral genome byproteinase K treatment. In order to degrade any contaminating DNA fromthe purified library, it was treated with DNase I enzyme (5 μl of 10U/μl) (Sigma-Aldrich; 4716728001) in 100 μl of DNase I buffer andincubated for 1 hat 37° C. The enzyme was inactivated by adding 5 μl of0.5 M EDTA at 70° C. for 10 min. Following DNase I treatment, the capsidprotein shell was digested by adding 120 μl of proteinase solutioncontaining 5 μl of 20 μg/μl of proteinase K and incubated at 50° C.overnight. To inactivate the proteinase K, the mixture was boiled at 95°C. The extracted rAAV library DNA was then concentrated and purifiedusing phenol chloroform and ethanol. An equal volume ofPhenol:Chloroform:Isoamyl Alcohol 25:24:1, pH8.0 (˜250 μl; ThermoFisherScientific; 15593031) was added and vortexed for 30 s. The mixture isincubated for 5 min at room temperature (RT) before centrifugation at15,000 rpm for 10 min at 4° C. The upper aqueous phase was separated andmixed with an equal volume of chloroform and vortexed for 30 s.Following 5 min incubation at RT, centrifuge at 15,000 rpm for 10 min at4° C. The upper aqueous phase was separated and one-tenth volume of 3Msodium acetate (pH 5.2) along with 2 μl Co-Precipitant Pink (Bioline;BIO-37075) and 2.5 volumes of ice cold 100% ethanol was added beforevortexing for 30 s. The mixture was incubated for at least 1 hr at ˜20°C. before centrifugation at 15,000 rpm for 15 min at 4° C. The pelletwas air dried and resuspended in TE buffer. The DNA concentration wasdetermined using the Qubit ssDNA assay.

Animals

All animal procedures performed in this study were approved by theCalifornia Institute of Technology Institutional Animal Care and UseCommittee (IACUC). C57BL/6J (000664), Tek-Cre (8863), SNAP25-Cre(23525), GFAP-Cre (012886), Synl-Cre (3966), and Ai14 (007908) micelines used in this study were purchased from the Jackson Laboratory(JAX). For in vivo library selection, 6- to 8-week-old adult male andfemale mice were intravenously injected with the viral libraries. Bothgenders were used for capsid selection to recover capsid variants withminimal gender bias. The IV injection of rAAVs was into theretro-orbital sinus of adult mice. For testing the transductionphenotypes of rAAVs, 6- to 8-week-old C57BL/6J or Tek-Cre or Ai14 adultmale mice were randomly assigned. The experimenter was not blinded forany of the experiments performed in this study.

In Vivo Selection

The 7-mer-i viral library selections were carried out in different linesof Cre transgenic adult mice: Tek-Cre, SNAP25-Cre, and GFAP-Cre for theR1 selections, and those three plus Syn1-Cre for the R2 selections. Maleand female adult mice were intravenously administered with a viralvector dose of 2×10¹¹ vg/mouse for the R1 selection, and a dose of1×10¹² vg/mouse for the R2 selection. The dose was determined based onthe virus yield which was different across selection rounds (FIG. 45).Both genders were used to recover capsid variants with minimal genderbias. Two weeks post-injection, mice were euthanized and all organsincluding brain were collected, snap frozen on dry ice, and stored at−80° C.

rAAV Genome Extraction from Tissue

Optimization. For rAAV genome extraction from tissues, both the Trizolmethod (Life Technologies; 15596) and the QlAprep Spin Miniprep kit(Qiagen, Inc; 27104) were used according to the manufacturers'recommended protocols, and found the Trizol method to be more efficient.The total rAAV genome recovery from 0.1 g of mouse liver was quantifiedby quantitative PCR using the primers mNeonGreen-F:5′-CGACACATGAGTTACACATCTTTGGCTC-3′ and mNeonGreen-R:5′-GGAGGTCACCCTTGGTGGACTTC-3′, which binds to the mNeonGreen gene of thessAAV-ACap-in-cis-Lox2 genome. As an internal control, the amount ofmitochondrial DNA (a measure of the recovery of smaller genomes) wasquantified using primers Mito-F: 5′-CCCAGCTACTACCATCATTCAAGT-3′ andMito-R: 5′-GATGGTTTGGGAGATTGGTTGATGT-3′. Although the percentage ofviral DNA per 1 ng total extracted DNA was about 1.5 fold higher withthe QlAprep kit than with the Trizol method, the overall recovery waslower with the QlAprep kit.

The extracted viral genome was digested with a restriction enzyme, suchas SmaI (found within the ITRs), to improve rAAV genome recovery by PCR.This was analyzed by quantitative PCR with Cre+primers, CapF-56:5′-ATTGGCACCAGATACCTG ACTCGTAA-3′, Cre+R-58:5′-CAAGTAAAACCTCTACAAATGTGGTAAAATCG-3′ and Cre-primers, CapF-56 (seeabove) and Cre-R-57: 5′-GTCCAAACTCATCAATGTATCTTATCATGTCTG-3′.

rAAV genome extraction with the Trizol method. Half of a frozen brainhemisphere (0.3 g approx.) was homogenized with a 2 ml glass homogenizer(Sigma Aldrich; D8938) or a motorized plastic pestle (FisherScientific;12-141-361, 12-141-363) (for smaller tissues) and processedas described in prior work. The extracted DNA was then treated with 3-6μl of 10 μg/μl RNase Cocktail Enzyme Mix (ThermoFisher Scientific;AM2286) to remove RNA and digested with Smal restriction enzyme. Thetreated mixture was then purified with a Zymo DNA Clean and Concentratorkit (D4033). From deep sequencing data analysis, it was observed thatthe amount of tissue processed for rAAV genome recovery is sufficient.

rAAV genome recovery by Cre-dependent PCR. rAAV genomes with Lox sitesflipped by Cre recombination were selectively recovered and amplifiedusing PCR with primers that yield a PCR product only if the Lox sitesare flipped (See FIG. 37). The primers 71F:5′-CTTCCAGTTCAGCTACGAGTTTGAGAAC-3′ and CDF/R:5′-CAAGTAAAACCTCTACAAATGTGGTAAAATCG-3′ were used and amplified theCre-recombined genomes over 25 cycles of 98° C. for 10 s, 58° C. for 30s, and 72° C. for 1 min, using Q5 DNA polymerase

Total rAAV genome recovery by PCR (Cre-independent). To recover all rAAVgenomes from a tissue, the primers XF(5′-ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC-3′) and 588-R2lib-R(5′-GTATTCCTTGGTTTTGAACCCAACCG-3′) were used to amplify the genomes over25 cycles of 98° C. for 10 s, 60° C. for 30 s, and 72° C. for 30 min,using Q5 DNA polymerase.

Sample Preparation for NGS

To analyze selections using deep sequencing, the DNA library wasprocessed, the virus library, and the tissue libraries post-in vivoselection to add flow cell adaptors around the diversified 7-merinsertion region (See FIG. 37).

Preparation of rAAV DNA and Viral DNA library. The Gibson-assembled rAAVDNA library and the DNA extracted from the viral library were amplifiedby Q5 DNA polymerase using the primers 588i-lib-PCR1-6bpUID-F:5′-CACGACGCTCTTCCGATCTAANNNNNNAGTCCTATGGACAAGTGGCCACA-3′ and588i-lib-PCR1-R:5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTGGTTTTGAACCCAACCG-3′ that arepositioned around 50 bases from the randomized 7-mer insertion on thecapsid, and that contain the Read1 and Read2 flow cell sequences on the5′ end.

The primer, 588i-lib-PCR1-6bpUID-F: 5′CACGACGCTCTTCCGATCTAANNNNNNAGTCCTATGGACAAGTGGCCACA-3′ used to minimallyamplify DNA and virus libraries for NGS has 6 nucleotides long UID(unique identifier) “NNNNNN” that sits after 19 nucleotides of Read-1sequence used in NGS “5′-CACGACGCTCTTCCGATCT”and linker “AA”. Thesequence after UID “AGTCCTATGGACAAGTGGCCACA” is the region that annealsto the AAV9 capsid. UID is an optional feature for NGS data analysis toidentify potential PCR amplification errors. However, this featurewasn't utilized in the NGS data analysis in this study to maintainconsistency with the primers used in rAAV genome recovery from tissueswhich lacks this UID feature (primers 71F: 5′-CTTCCAGTTCAGCTACGAGTTIG.AGAAC-3 and CDF/R: 5′ CAAGTAAAACCTCT ACA AATGTGGTAA AATCG-3). The UID or any kind of overhangs seemed to affectthe PCR based recovery from tissue. Presumably, the primerthermostability has a key role to play in very low amount of extractedrAAV genomes from tissues.

Using 5-10 ng of template DNA in a 50 μl reaction, the DNA was minimallyamplified for 4 cycles of 98° C. for 10 s, 60° C. for 30 s, and 72° C.for 10 s. The mixture was then purified with a PCR purification kit. Theeluted DNA was then used as a template in a second PCR to add the uniqueindices (single or dual) via the recommended primers (NEB; E7335S,E7500S, E7600S) in a 12-cycle reaction using the same temperature cycleas described above. The samples were then sent for deep sequencingfollowing additional processing and validation.

The PCR products post indices addition were run on a freshly prepared 2%low-melting-point agarose gel (ThermoFisher Scientific; 16520050) forbetter separation and recovery of the approx. 120 bp DNA band on thegel. Before sending the sample for NGS, the nucleotide diversity at therandomized 7-mer position was verified by Sanger sequencing. If needed,an optional PCR was carried out to send sufficient sample for Sangersequencing using 15-20 cycles of 98° C. for 10 s, 60° C. for 30 s, and72° C. for 10 s with the primers NGS-QC-F: 5′-AATGATACGGCGACCACCGAG-3′and NGS-QC-R: 5′-CAAGCAGAAGACGGCATACGA-3′. Upon validation, thelibraries were sent for deep sequencing using the Illumina HiSeq 2500System (Millard and Muriel Jacobs Genetics and Genomics Laboratory,Caltech; Integrative Genomics Core, City of Hope).

Preparation of rAAV tissue DNA library. The PCR-amplified rAAV DNAlibrary from tissue (see section: In vivo selection (i) (c)) was furtheramplified with a 1:100 dilution of this DNA as a template to the primers1527: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGACAAGTGGCCACAAACCACCAG-3′ and1532: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTTGGTTTTGAACCCAACCG-3′ thatare positioned around 50 bases from the randomized 7-mer insertion onthe capsid, and that contain the Read1 and Read2 sequences on the 5′end. The DNA was amplified by Q5 Hot Start High-Fidelity 2X Master Mixor NEBNext Ultra II Q5 Master Mix (NEB; M0544) for 10 cycles of 98° C.for 10 s, 59° C. for 30 s, and 72° C. for 10 s. The mixture was purifiedwith a PCR purification kit. The eluted DNA was then used as a templatein a second PCR to add the unique indices (single or dual) using therecommended primers (NEB; E7335S, E7500S, E7600S) in a 10-cycle reactionwith the same temperature cycle as described above (for DNA and viruslibrary preparation). The extracted DNA was validated by Sangersequencing and sent for deep sequencing as described in the previoussection.

In Vivo Characterization of AAV Vectors

Cloning AAV capsid variants. The AAV capsid variants were cloned into apUCmini-iCAP-PHP.B backbone (Addgene ID: 103002) using overlappingforward and reverse primers with 11-mer substitution (in case of 7-mer-ivariants, the flanking amino acids from AAV9 capsid AA587-588 “AQ” andAA589-590 “AQ” were subjected to codon modification) that spans from theMscl site (at position 581 AA) to the Agel site (at position 600 AA) onthe pUCmini plasmid. The primers were designed for all capsid variantsusing a custom python script (Code will be made available on github),and since they cover the entire fragment insertion, these primers areself-annealed and amplified using PCR to create a dsDNA fragment withoutthe use of a template DNA. They are amplified by Q5 Hot StartHigh-Fidelity 2X Master Mix for 20 cycles of 98° C. for 10 s, 60° C. for30 s, and 72° C. for 10 s. This fragment was then assembled into theMscI/AgeI digested pUCmini-iCAP-PHP.B backbone by the Gibson assemblymethod. There is a second Mscl site on the backbone; however, this wasblocked by methylation. The assembled plasmids were then transformedinto NEB Stable Competent E. coli (New England Biolabs, Inc; C3040H),and colonies were selected on carbenicillin/ampicillin-LB agar plates.

List of primers used to clone AAV-PHP variants: The variants from7-mer-i and 3-mer-s libraries were cloned as 11-mer substitution.

TABLE 6 Primers For AAV-PHP Variants Variant Amino NucleotideForward primer Name acid sequence (5′-3′) Reverse primer (5′-3′) AAV-AQTALKP GCCCAAA GGAGTCCTATGGAC TTCCTTGGTTTTGAAC PHP.V1 FLAQ CCGCCCTCAAGTGGCCACAAA CCAACCGGTCTGCGC AAACCCTT CCACCAGAGTGCCC CTGTGCGAGGAAGGGCCTCGCAC AAACCGCCCTCAAA TTTGAGGGCGGTTTGG AG CCC (SEQ ID NO:GC (SEQ ID NO: 1047) 1032) AAV- AQTTLKP GCCCAAA GGAGTCCTATGGACTTCCTTGGTTTTGAAC PHP.V2 FLAQ CCACCCTC AAGTGGCCACAAA CCAACCGGTCTGCGCAAACCCTT CCACCAGAGTGCCC CTGTGCGAGGAAGGG CCTCGCAC AAACCACCCTCAAATTTGAGGGTGGTTTGG AG CCC (SEQ ID NO: GC (SEQ ID NO: 1048) 1033) AAV-AQTLQIPF GCCCAAA GGAGTCCTATGGAC TTCCTTGGTTTTGAAC PHP.B4 KAQ CGTTGCAGAAGTGGCCACAAA CCAACCGGTCTGCGC ATTCCTTT CCACCAGAGTGCCC CTGTGCCTTAAAAGGTAAGGCA AAACGTTGCAGATT AATCTGCAACGTTTGG CAG CCT (SEQ ID NO:GC (SEQ ID NO: 1049) 1034) AAV- AQTLQLP GCCCAAA GGAGTCCTATGGACTTCCTTGGTTTTGAAC PHP.B5 FKAQ CCCTCCAA AAGTGGCCACAAA CCAACCGGTCTGCGCT(SEQ ID CTCCCCTT CCACCAGAGTGCCC TGGGCTTTGAAGGGG NO: 1062) CAAAGCCAAACCCTCCAACTC AGTTGGAGGGTTTGG CAA (SEQ CCC (SEQ ID NO:GC (SEQ ID NO: 1050) ID NO: 1035) 1068) AAV- AQTLQQP GCCCAAAGGAGTCCTATGGAC TTCCTTGGTTTTGAAC PHP.B6 FKAQ CTTTGCAG AAGTGGCCACAAACCAACCGGTCTGCGC (SEQ ID CAGCCGTT CCACCAGAGTGCCC CTGTGCCTTAAACGGCNO: 1063) TAAGGCA AAACTTTGCAGCAG TGCTGCAAAGTTTGG CAG (SEQCCG (SEQ ID NO: GC (SEQ ID NO: 1051) ID NO: 1036) 1069) AAV- AQSIERPFGCCCAAA GGAGTCCTATGGAC TTCCTTGGTTTTGAAC PHP.B7 KAQ GCATCGA AAGTGGCCACAAACCAACCGGTCTGCGC AAGACCCT CCACCAGAGTGCCC CTGTGCTTTGAAGGGT TCAAAGCAAAGCATCGAAAG CTTTCGATGCTTTGGG ACAG ACCC (SEQ ID NO: C (SEQ ID NO: 1052)1037) AAV- AQTMQKP GCCCAAA GGAGTCCTATGGAC TTCCTTGGTTTTGAAC PHP.B8 FIAQCCATGCAA AAGTGGCCACAAA CCAACCGGTCTGCGC AAACCCTT CCACCAGAGTGCCCCTGTGCGATGAAGGG CATCGCAC AAACCATGCAAAA TTTTTGCATGGTTTGG AGACCC (SEQ ID NO: GC (SEQ ID NO: 1053) 1038) AAV- AQRYQGD GCCCAAAGGAGTCCTATGGAC TTCCTTGGTTTTGAAC PHP.C1 SVAQ GGTATCAG AAGTGGCCACAAACCAACCGGTCTGCGC GGTGATTC CCACCAGAGTGCCC CTGTGCAACAGAATC TGTTGCACAAAGGTATCAGGG ACCCTGATACCTTTGG AG TGAT (SEQ ID NO: GC (SEQ ID NO: 1054)1039) AAV- AQWSTNA GCCCAAT GGAGTCCTATGGA TTCCTTGGTTTTGAA PHP.C2 GYAQGGTCGAC CAAGTGGCCACAA CCCAACCGGTCTGCG AAACGCT ACCACCAGAGTGCCCTGTGCGTAACCAG GGTTACG CCAATGGTCGACA CGTTTGTCGACCATT CACAGAACGCT (SEQ ID GGGC (SEQ ID NO: NO: 1040) 1055) AAV- AQERVGF GCCCAAGGGAGTCCTATGGA TTCCTTGGTTTTGAA PHP.C3 AQAQ AGCGTGT CAAGTGGCCACAACCCAACCGGTCTGCG AGGTTTC ACCACCAGAGTGC CCTGTGCCTGTGCGA GCACAGGCCAAGAGCGTGTA AACCTACACGCTCTT CACAG GGTTTC (SEQ ID GGGC (SEQ ID NO:NO: 1041) 1056) AAV-PHP.N AQTLAVP GCGCAGA GGAGTCCTATGGACTTCCTTGGTTTTGAAC FSNP (SEQ CCCTAGCT AAGTGGCCACAAA CCAACCGGTCTGCGC ID NO:GTCCCTTT CCACCAGAGTGCGC AGGGTTCGAAAAAGG 1064) TTCGAACC AGACCCTAGCTGTCGACAGCTAGGGTCTG CT (SEQ ID CCT (SEQ ID NO: CGC (SEQ ID NO: 1057)NO: 1070) 1042) AAV- AQARQM GCCCAAG GGAGTCCTATGGAC TTCCTTGGTTTTGAACPHP.X1 DLSAQ CCAGACA AAGTGGCCACAAA CCAACCGGTCTGCGC (SEQ ID AATGGACCCACCAGAGTGCCC CTGTGCGCTGAGGTCC NO: 1065) CTCAGCGC AAGCCAGACAAATATTTGTCTGGCTTGGG ACAG (SEQ GGAC (SEQ ID NO: C (SEQ ID NO: 1058) ID NO:1043) 1071) AAV- AQTNKVG GCCCAAA GGAGTCCTATGGAC TTCCTTGGTTTTGAAC PHP.X2NIAQ (SEQ CCAACAA AAGTGGCCACAAA CCAACCGGTCTGCGC ID NO: AGTCGGCCCACCAGAGTGCCC CTGTGCGATGTTGCCG 1066) AACATCGC AAACCAACAAAGTACTTTGTTGGTTTGGG ACAG (SEQ CGGC (SEQ ID NO: C (SEQ ID NO: 1059) ID NO:1044) 1072) AAV- AQQNVTK GCCCAAC GGAGTCCTATGGA TTCCTTGGTTTTGAA PHP.X3GVAQ AGAACGT CAAGTGGCCACAA CCCAACCGGTCTGCG AACGAAG ACCACCAGAGTGCCCTGTGCCACACCCT GGTGTGG CCAACAGAACGTA TCGTTACGTTCTGTT CACAGACGAAG (SEQ ID GGGC (SEQ ID NO: NO: 1045) 1060) AAV- AQLNAIK GCCCAACGGAGTCCTATGGA TTCCTTGGTTTTGAA PHP.X4 NIAQ (SEQ TCAACGC CAAGTGGCCACAACCCAACCGGTCTGCG ID NO: TATCAAG ACCACCAGAGTGC CCTGTGCGATGTTCT 1067)AACATCG CCAACTCAACGCT TGATAGCGTTGAGTT CACAG ATCAAG (SEQ IDGGGC (SEQ ID NO: (SEQ ID NO: 1046) 1061) NO: 1073)

AAV vector production. Using an optimized protocol, AAV vectors wereproduced from 5-10 150 mm plates, which yielded sufficient amounts foradministration to adult mice.

AAV vector administration. AAV vectors were administered intravenouslyto adult male mice (6-8 weeks of age) via retro-orbital injection atdoses of 1-10×10¹¹ vg. The AAV doses are determined by the experimentalneeds. CAG-NLS-GFP related experiments for quantification were done atmedium dose of 1×10¹¹ vg given this was the dose previously determinedfor AAV-PHP.eB characterization. Otherwise, the non-NLS genome relatedexperiments were done at 3×10¹¹ vg, with the exception of Cre-driverlines (GFAP-Cre or Tek-Cre), or a lower strength promoter containinggenome (GFAP-NLS-mTurq) where the dose was 1×10¹² vg. The high dose waschosen to understand the full potential of the new vectors in thesesystems.

All experiments with vectors carrying CAG, a strong ubiquitous promoter,were incubated for 3 weeks. The 4 week incubations are those thatinvolved expression from Cre driver lines or cell-type specific promoterwhere it is generally recommended for a longer wait time. The 2 weekincubations are those where the vectors carried self-complementarygenomes with strong ubiquitous promoters.

Tissue processing. After 3 weeks of expression (unless noted otherwise),the mice were anesthetized with Euthasol (pentobarbital sodium andphenytoin sodium solution, Virbac AH) and transcardially perfused with30-50 mL of 0.1 M phosphate buffered saline (PBS) (pH 7.4), followed by30-50 ml of 4% paraformaldehyde (PFA) in 0.1 M PBS. After thisprocedure, all organs were harvested and post-fixed in 4% PFA at 4° C.overnight. The tissues were then washed and stored at 4° C. in 0.1 M PBSand 0.05% sodium azide. All solutions used for this procedure werefreshly prepared. For the brain and liver, 100-_(i).tm thick sectionswere cut on a Leica VT1200 vibratome.

For vascular labeling, the mice were anesthetized and transcardiallyperfused with 20 mL of ice-cold PBS, followed by 10 mL of ice-cold PBScontaining Texas Red-labeled Lycopersicon Esculentum (Tomato) Lectin(1:100, Vector laboratories, TL-1176), and then placed in 30 mL ofice-cold 4% PFA for fixation.

Immunohistochemistry. Tissue sections—typically 100-μm thick—were firstincubated in blocking buffer (10% normal donkey serum, 0.1% TritonX-100, and 0.01% sodium azide in 0.1 M PBS, pH 7.4) with primaryantibodies at appropriate dilutions for 24 h at room temperature on arocker. The primary antibodies used in this study were rabbit S100(1:400, Abcam, ab868), rabbit Olig2 (1:400; Abcam, ab109186), rabbitNeuN (1:400, Abcam, ab177487), and rabbit GLUT-1 (1:400; MilliporeSigma, 07-1401). After primary antibody incubation, the tissues werewashed 1-3 times with wash buffer 1 (0.1% Triton X-100 in 0.1 M PBSbuffer, pH 7.4) over a period of 5-6 h in total. The tissues were thenincubated in blocking buffer with the secondary antibodies atappropriate dilutions for 12-24 h at room temperature and then washed inthree times in 0.1 M PBS, pH 7.4 over a total duration of 5-6 h. Thesecondary antibody used in this study was Alexa Fluor 647 AffiniPuredonkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch Lab, 711-605-152).When performing nuclear staining, 4′,6-Diamidine-2′-phenylindoledihydrochloride (DAPI, Sigma Aldrich, 10236276001) is used at a 1:1000dilution in 0.1 M PBS, pH 7.4 and incubated with tissues for 15 minutesfollowed by a single wash for 10 minutes in 0.1 M PBS, pH 7.4. The DAPIand/or antibody-stained tissue sections were mounted with ProLongDiamond Antifade Mountant (ThermoFisher Scientific, P36970).

Hybridization chain reaction (HCR) based RNA labeling in tissues.Fluorescence in situ hybridization chain reaction (FITC-HCR) was used tolabel excitatory neurons with VGLUT1 and inhibitory neurons with GAD1 tocharacterize the AAV capsid variant AAV-PHP.N in brain tissue using anadapted third-generation HCR protocol. To characterize the AAV capsidvariant AAV-PHP.N in brain tissue, HCR method was sought to labelexcitatory and inhibitory neurons. Fluorescence in situ hybridizationchain reaction (FITC-HCR) was used to label excitatory neurons withVGLUT1 and inhibitory neurons with GAD1. Adapting the third-generationHCR, 13 probe sets were designed for each target by using custom-madesoftware (https://github.com/GradinaruLab/HCRprobe). After 3 weeks ofexpression, the mice were transcardially perfused and fixed as describedearlier (Section D. Tissue processing). To minimize RNase enzymeexposure in fixed tissues, following overnight fixation in 4% PFA, thetissues were washed and stored at 4° C. in 0.1 M RNase-free PBS and0.05% sodium azide. The harvested brains were henceforth handled withcare to avoid exposure to RNase using reagents such as RNAlaterstabilization solution/RNase-free PBS/ RNaseZap (ThermoFisherScientific, AM7021, AM9624, AM9780). Once the harvested brains weresagittally sliced to 100-pm thick sections, FITC-HCR was performed todetect both genes. Tissue slices were permeabilized with 0.1% TritonX-100 in 0.1 M RNase-free PBS for 1 h at RT and pre-hybridized inhybridization solution (10% dextran sulfate and 10% ethylene carbonatein 2xSSC buffer (saline-sodium citrate)) for >30 min at 37° C. Thedesigned probes were diluted in hybridization solution to get a finalconcentration of 2 nM. The tissue sections were then subjected tohybridization with the probes overnight at 37° C. Following this, thesections were washed with pre-warmed wash buffer (10% ethylene carbonatein 2xSSC) at 37° C. for 30 min twice, followed by 2xSSC at RT for 30 mintwice. Amplification with hairpin pairs (Molecular Technologies, CA)were performed in amplification buffer (10x dextran sulfate in 2xSSC);hairpins were snap-cooled at 95° C. for 90 s, followed by RT for 30 min,and diluted with amplification buffer (60 nM). Tissues were thenincubated in this amplification buffer with hairpins overnight at RTwith gentle agitation. Once the amplification was done, samples werebriefly washed with 2xSSC and mounted in Prolong Diamond for imaging.

Imaging and image processing. All images in this study were acquiredeither with a Zeiss LSM 880 confocal microscope using the objectivesFluar 5×0.25 M27, Plan-Apochromat 10×0.45 M27 (working distance 2.0 mm),and Plan-Apochromat 25×0.8 Imm Con DIC M27 multi-immersion; or with aKeyence BZ-X700 microscope. The acquired images were processed in ZenBlack 2.3 SP1 (Zeiss), BZ-X Analyzer (Keyence), Illustrator CC 2018(Adobe), Photoshop CC 2018 (Adobe), and Imaris (Bitplane). To preventany imaging artifacts resulting from multiple fluorescence spectraloverlap, the fluorescence excitation and emission spectra were keptdistinct following the recommended linear unmixed acquisition ofindividual colors. A far-red fluorescent dye was chosen for anyadditional marker staining to keep the imaging parameters distinct fromin vivo fluorescent expression thereby preventing any spectral overlapacross detector channels. The tissues were routinely monitored for autofluorescence or imaging artifacts before acquisition, and imagingparameters were adjusted if needed. The imaging parameters werecross-checked with tissues lacking in vivo transduction to avoid anyimaging artifacts. The regions used for the images were closely matchedacross experimental groups to minimize bias during comparisons.

Tissue clearing and imaging of thick tissues. To demonstrate the abilityof PHP.V1 to transduce the vasculature across thick tissues, such ashalf of a mouse-brain hemisphere or a femur bone, tissue from Tek-Cremice was assessed after 4 weeks' of expression.

The brain hemisphere was stained with the primary antibody, Anti-GFP(Ayes Labs, GFP-1020), and the secondary antibody, goat anti-ChickenIgY, Alexa Fluor 633 (ThermoFisher Scientific, A-21103), and cleared viathe iDISCO protoco1³⁸. For imaging, a commercial light-sheet microscope(Lavision BioTec) with a custom objective lens (4×) was used. Theresulting image files were reorganized by a custom MATLAB script toallow stitching with TeraStitcher. For 3-D visualization, Imaris(Bitplane) was used.

To image the mouse femur bone, the bones were sectioned to 300 μmthickness for antibody penetration and stained with the primaryantibody, Anti-GFP, and the secondary antibody, Alexa Fluor 488 donkeyanti-chicken IgY (Jackson ImmunoResearch Lab, 703-545-155), and thencleared via the TDE (2-2′-thiodiethanol) clearing method⁴¹. The imageswere acquired with a confocal microscope (Zeiss LSM 880) and visualizedin Imaris software.

Tissue processing and imaging for quantification of rAAV transduction invivo. For quantification of rAAV transduction, 6- to 8-week-old malemice were intravenously injected with the virus, which was allowed toexpress for 3 weeks (unless specified otherwise). The mice were randomlyassigned to groups and the experimenter was not blinded. The mice wereperfused and the organs were fixed in PFA. The brains and livers werecut into 100-μm thick sections and immunostained with differentcell-type-specific antibodies, as described above. The images wereacquired either with a 25× objective on a Zeiss LSM 880 confocalmicroscope or with a Keyence BZ-X700 microscope; images that arecompared directly across groups were acquired and processed with thesame microscope and settings.

For quantification of PHP.B-family variant transduction in tissues, theimages were acquired using 25x objective with lx digital zoom on a ZeissLSM 880 confocal microscope. With n=3 mice per variant, images wereacquired across 4 brain regions—cortex, striatum, ventral midbrain andthalamus, and tissues were stained with 3 cell type markers (NeuN,Olig2, and S100). For each mouse, 2 images per brain region per celltype marker were acquired, and the mean were plotted.

For PHP.N transduction analysis, the images were acquired using 20×objective on Keyence BZ-X700 microscope. With n=3 mice, images across 4brain regions—cortex, striatum, ventral midbrain and thalamus wereacquired to cover the entire brain regions for 3 cell type markers(NeuN, Olig2, and S100). This involved 6-8 images to cover cortex,thalamus and striatum, and 2 images to cover ventral midbrain per mouseper cell-type marker. For each mouse, across each region, the mean fromthe images were plotted.

For PHP.V GLUT1⁺transduction analysis, the images were acquired using25× objective with lx digital zoom on a Zeiss LSM 880 confocalmicroscope. Each distinct blood vessel in the image with GLUT1⁺stainingand XFP expression was determined as positive for transduction.Quantification of expression from the CAG-mNeonGreen vector wasperformed across the cortex (n=3 per group). Each data point is drawnfrom the mean of 3-2 images per mouse. Different brain regions werequantified for Tek-Cre and Ai14 mouse experiments with n=2 per group.For cortex, cerebellum, striatum and ventral midbrain, the mean wasplotted from 3-4 images per mouse per region.

In Vitro Characterization of AAV Vectors

Human Brain Microvascular Endothelial Cells (HBMEC) (ScienCell ResearchLaboratories, Cat. 1000) were cultured as per the instructions providedby the vendor. HBMEC were cultured from a frozen stock vial infibronectin-coated T-75 flask (7000-9000 cells/cm² seeding density)using the Endothelial Cell Medium (Cat. 1001). The cells weresubcultured in fibronectin-coated 48-well plates (0.95 cm² growth area)at the recommended seeding density and incubated at 37° C. for ˜24-48 htill the cells were completely adherent with -18 70-80% confluence. Theviral vectors packaging pAAV-CAG-mNeongreen were added to the cellculture at a dose of either 1×10⁸ or 1×10¹⁰ vg per well (3 wells perdose per vector). The media was changed 24 hours later and the culturewas assessed for fluorescence expression at 3 days' post infection. Pervendor recommendation, the culture media was changed every other day tomaintain the cell culture.

Data Analysis

Quantification of rAAV vector transduction in mouse tissue. Manualcounting was performed with the Adobe Photoshop CC 2018 Count Tool forcell types in which expression and/or antibody staining covered thewhole cell morphology. The Keyence Hybrid Cell Count software (BZ-H3C)was used where the software could reliably detect distinct cells in anentire dataset. To maintain consistency in counting across differentmarkers and groups, one person was assigned to quantify across allgroups in all brain areas.

Manual counting was performed for GLUT-1-stained blood vessels andexpression of the ssAAV:CAG-mNeonGreen and ssAAV:CAG-DIO-EYFP, where theefficiency was calculated as the percentage of XFP+vessels relative tothe GLUT-1 staining. Manual counting was also performed to quantifynuclear or soma stained cells, including NeuN-, Olig2-, and S100-stainedcells. The efficiency was calculated as the percentage of XFP+cellsrelative to cell-marker+cells.

Keyence Hybrid Cell Count software (BZ-H3C) was used to quantifyexpression of nuclear localized AAV genomes in liver hepatocytes thatco-localized with the DNA stain, DAPI; and also for the study involvingssAAV:GFAP-2xNLS-mTurquoise2 genomes with S100 cell marker.

The mean fluorescence intensity across microscopic images werequantified using ImageJ software. The images were processed forbackground subtraction and using the Threshold operation, the meanfluorescence intensity was measured.

NGS data alignment and processing. The raw fastq files from NGS runswere processed with custom built scripts (codes will be made availableon Github) that align the data to AAV9 template DNA fragment containingthe diversified region 7xNNK (for R1) or 11xNNN (for R2 since it wassynthesized as 11xNNN). The pipeline to process these datasets involvedfiltering the dataset to remove the low-quality reads by using the deepsequencing quality score for each sequence. The variant sequences werethen recovered from the sequencing reads by searching for the flankingtemplate sequences, and extracting the nucleotides of the diversifiedregion (perfect string match algorithm). The quality of the aligned datawas further investigated to remove any erroneous sequences (such as oneswith stop codons). The raw data was plotted (as shown in SupplementaryFIG. 1e ) to study the quality of recovery across every library. Basedon the RC distribution, we adapted a thresholding method to removeplausible erroneous mutants that may have resulted from PCR or NGS basederrors. The assumption is that if there is a PCR mutation or NGS erroron the recovered parent sequence, the parent must have existed at leastone round earlier than the erroneous sequence, and thus a difference inRCs should exist.

For R1 tissue libraries, a steep drop was observed in the slope of thedistribution curve following a long tail of low count sequences, andwere found to be rich in sequences that are variations of the parents inthe higher counts range. A threshold for RCs was manually set to removesuch erroneous mutants. The thresholded data were then processeddifferently based on the experimental needs as described elsewhere usingcustom Python based scripts.

For R2 tissue libraries from PCR pool and synthetic pool, given thesmaller library size compared to R1, the data was thresholded in twosteps. Only the tissue recovered sequences that were present in therespective input DNA and virus library were considered (after removinglower count variants from input libraries following the same principleas R1 tissue libraries). This step partially removed the long tail oflow count reads. As a second step, the thresholding that was describedfor R1 tissue libraries was applied.

While it is plausible that true variants may be lost duringthresholding, this method minimized false positives as the low countmutants in tissue and virus libraries often seemed to have very highenrichment score (as RCs are normalized to input library). In otherwords, thresholding allowed selective investigation on positively andnegatively enriched variants that had a higher-confidence in their NGSRCs.

As an alternative to the manual thresholding method, an optional errorcorrection method called “Collapsing” was built to further validate theoutcome from filtered datasets. This method starts at the lowest countvariants (variants of count 1) and searches for potential parentvariants that are off by one nucleotide but have at least 2-fold highercounts (fold change=(2^(ΔCT)) where CT is PCR cycle threshold). Thiserror correction method then transfers the counts of these potentialerroneous sequences to their originating sequences and repeatsrecursively until all sequences have been considered. On applying thiserror correction to the thresholded data, an additional ˜0.002-0.03% ofsequences were captured (compared to >19% captured by thresholding),confirming that the thresholding strategy was largely successful.

NGS data analysis. The aligned data were then further processed via acustom data-processing pipeline, with scripts written in Python(available on Github). The enrichment scores of variants (Total =N)across different libraries were calculated from the read counts (RCs)according to the following formula: Enrichment score =log10 [(Variant IRC in tissue libraryl/ Sum of variants N RC in libraryl)/(Variant I RCin virus library/ Sum of variants N RC in virus library)]

To consistently represent library recovery between R1 and R2 selectedvariants, the enrichment score of the variants in R1 selection wasestimated. The standard score of variants in a specific library wascalculated using this formula: Standard score=(readcount_i-mean)/standard deviation. Where read count_i is raw copy numberof a variant i, Mean is the mean of read counts of all variants across aspecific library, Standard deviation is the standard deviation of readcounts of all variants across a specific library.

Since the DNA and virus libraries were not completely sampled unlike thetissue libraries, we assigned an estimated RC for variants that were notpresent in the input library but were present in the output library. Forinstance, R1 virus library is the input library to the R1 tissuelibraries. The estimated RC is defined as a number that is lower thanthe lowest RC in the library with the assumption that these variantswere found at a relatively lower abundance than the variants recoveredfrom the deep sequencing. In virus libraries, since RC of 1.0 was thelowest, we assigned all missing variants an estimated RC of 0.9. We usethis method to calculate the enrichment score of the R1 tissue librarieswhich is normalized to R1 virus library (FIG. 1d ). This was done torepresent libraries across two selection rounds consistently. Although,the individual enrichment score among R1 variants didn't add asignificant value to the variants selected for R2 selection as describedin the criteria to separate signal vs noise in R1 using the RCs.

Heatmap generation. The relative AA distributions of the diversifiedregions are plotted as heatmaps. The plots were generated using thePython Plotly plotting library. The heatmap values were generated fromcustom scripts written in Python, using functions in the custom “pepars”Python package. Each heatmap uses both an expected (input) distributionof amino acid sequences and an output distribution. The outputdistribution must be a list of sequences and their count, and the inputdistribution can be either a list of sequences and their count, or anexpected amino acid frequency from a template, such as NNK. For bothinput and output, the total count of amino acids in each position istallied in accordance to each sequence's count and then divided by thetotal sum of counts, giving a frequency of each amino acid at eachposition. Then, the log2 fold change is calculated between the outputand the input. For amino acids with a count of 0 in either the input oroutput, no calculation is performed. In order to distinguish betweenstatistically significant amino acid biases, a statistical test wasperformed using the statsmodels Python library. For the case where thereare two amino acid counts, a two-sided, two-proportion z-test wasperformed; for comparing the output amino acid count to an expectedinput frequency from a template, a one-proportion z-test was performed.All p-values were then corrected for multiple comparisons usingBonferroni correction. Only bias differences below a significancethreshold of le-4 are then outlined on the heatmap; all other(insignificant) squares are left empty.

Clustering analysis. Using custom scripts written in MATLAB (versionR2017b; MathWorks) the reverse Hamming distances representing the numberof shared AAs between two peptides was determined. Cytoscape (version3.7.1⁵³) software was then used to cluster the variants. The AAfrequency plot representing the highlighted cluster was created usingWeblogo (Version 2.8.2). The reverse Hamming distances (representing thenumber of shared AAs between two peptides) was determined for all uniquecapsid variants with greater than 10 count and greater than 2.5-foldenrichment after R2 selection. This process iteratively compares eachvariant with all other variants within the group. Capsid variants werethen clustered by their reverse Hamming distances using Cytoscape. Theminimum reverse Hamming distance for visualization was chosen manuallybased on sequence similarity.

For the amino acid frequency plots, the number on the bottom representsthe position of the diversified motif starting from 1. The size of theamino acid in the stack reflects the proportion of unique clones inwhich the AA appears at that specific position in the motif. The colorcode is based on the AA properties. The positively charged residues K,R, and H are in blue. The negatively charged residues D and E are inred. The amide containing polar residues Q, and N are in magenta. Thepolar residues T, and S, are in green. The hydrophobic residues A, L, V,I, P, F, M, and W are in black.

Example 3 Multiplexed-CREATE Allows Detailed Characterization of theCapsid Libraries During Round-1 Selection

To identify variants that enrich in specific cell types or organsparallel selections across multiple targets were performed, and theenrichment or depletion of each capsid variant across those targets wasmapped.

During DNA and virus library generation there is potential foraccumulation of biases that over-represent certain capsid variants,obscuring their true enrichment during in vivo selection. These biasesmay result from PCR amplification bias in the DNA library or sequencebias in the efficiency of virus production across various steps: capsidassembly, genome packaging and stability during purification. This wasinvestigated with the 7-mer-i library, a randomized 7-mer libraryinserted between positions 588-589 of AAV9 (FIGS. 1 and 2) inrAAV-ACap9-in-cis-Lox2 plasmid (FIG. 36). Sequencing libraries after DNAassembly and virus purification to a depth of 10-20 million (M) readswas adequate to capture the bias among variants during virus production(FIG. 3; despite ˜1% variant overlap among these libraries; FIGS. 38 and39), demonstrating that even permissive sites like 588-589 will imposebiological constraints on sampled sequence space. The DNA library had auniform distribution of 9.6 M unique variants within ˜10 M total reads(read count (RC) mean=1.0, S.D. =0.074), indicating minimal bias. Incontrast, the virus library had 3.6 M unique variants within ˜20 M depth(RC mean=4.59, S.D. =11.15) indicating enrichment of a subset ofvariants during viral production.

For in vivo selection, the 7-mer-i viral library was intravenouslyinjected at a dose of 2×10¹¹ vg per adult transgenic mouse expressingCre in different brain cell types: GFAP-Cre mice for astrocytes,SNAP25-Cre mice for neurons, and Tek-Cre mice for endothelial cells (n=2mice per Cre transgenic line, see Methods). Two weeks after intravenous(IV) injection, the brain and liver tissues were harvested, with thelatter serving as a control organ since AAV9 transduces it with highefficiency. The rAAV genomes were extracted from tissues and the capsidsthat transduced Cre-expressing cells were selectively amplified (FIGS.40-44). Upon deep sequencing, ˜8×10⁴ unique nucleotide variantsrecovered from brain tissues and <50 variants in spinal cords (˜48% ofwhich were identified in virus library) were observed across thetransgenic lines, and each variant was represented with an enrichmentscore reflecting the

Design Parameters Synthetic pool design PCR pool designchange in relative abundance between the brain and the starting viruslibrary (FIG. 4).

Two features of this dataset stand out. First, the recovered variants inbrain tissue were disproportionately represented among the fraction ofthe transformed capsid library observed by sequencing after viralproduction demonstrating how production biases skew selection results.Second, the distribution of capsid read counts (RCs) revealed that morethan half of the unique recovered variants after selection appear atremarkably low read counts. These variants may either be unintendedmutants from experimental manipulation or AAV9-like variants with lowbasal level of CNS transduction (FIG. 40).

Example 4 A Novel Round-2 Library Design Improves the Selection Outcome

Concerned that the sequence bias during viral production and recoverywould propagate across selection rounds despite post-hoc enrichmentscoring, an unbiased library was designed based on the round-1 (R1)output (synthetic pool library) via oligo pools (Twist Bioscience). Thislibrary was compared to a library PCR amplified directly from therecovered R1 DNA (PCR pool library) (FIG. 5, Table 7).

Table 7: Comparison between the two methods for R2 selection. The tablesummarizes the pros and cons of selection design parameters by thesynthetic pool and PCR pool R2 selection methods.

Carryover of No, likelihood Yes, potential R1 selection of false tominimize bias among variants positives is low by normalization Carryoverof R1 selection No Yes induced mutants Confidence in library High, usingalternate Low performance codon replicates Customize library or add Yes,in an Yes, with greater internal controls unbiased manner risk of biasControl library size Yes, without reducing Yes, with libraries librariesor pooling reduced for pooling Cost for R2 library High Low generation

The synthetic pool library design comprised: (1) equimolar amounts of˜8950 capsid variants present at high read counts in at least one of theR1 selections from brain and spinal cord (FIG. 40); (2) alternativecodon replicates of those ˜8950 variants (optimized for mammaliancodons) to reduce false positives; and (3) a “spike-in” library ofcontrols (FIGS. 74 and 75), resulting in a total library size of 18,000nucleotide variants.

As anticipated, both round-2 (R2) virus libraries produced a high titer(˜6×10¹¹ vg per 10 ng of R2 DNA library per 150 mm dish; FIGS. 45), and˜99% of variants from the R2 DNA were found after viral production (FIG.6). However, the distribution of the DNA and virus libraries from bothdesigns differed significantly. The PCR pool library carries forward theR1 selection biases (FIGS. 7, 46, and 47) where the abundance reflectsprior enrichment across tissues in R1 as well as bias from viralproduction and sample mixing. Comparatively, the synthetic pool DNAlibrary is more evenly distributed, minimizing bias amplification acrossselection rounds.

For in vivo selection, a dose of 1×10¹² vg per adult transgenic mousewas administered into three of the previously used lines (n=2 mice perCre transgenic line—GFAP, SNAP25, Tek), as well as the Syn-Cre line (forneurons). Two weeks after IV injection, rAAV genomes from brain sampleswere extracted, selectively amplified, and deep sequenced (as in R1).The synthetic pool library produced a greater number of positivelyenriched capsid variants than the PCR pool brain library (e.g. ˜1700versus ˜700 variants/tissue library at amino acid (AA) level inGFAP-Cre) (FIGS. 8 and 48). In the synthetic pool, ˜90% of the variantsfrom the spike-in library were positively enriched as expected (FIG. 48,middle panel; FIG. 74).

The degree of correlation for enrichment scores of variants recoveredfrom both PCR and synthetic pool libraries varies in each Cre transgenicline, demonstrating the presence of noise within experiments (FIG. 49).The synthetic pool's codon replicate feature addresses this predicamentby pinpointing the level of enrichment needed within each selection torise above noise (FIGS. 9, 50, and 51). This is a significant advantageover the PCR pool design, allowing researchers to confidently interpretenrichment scores in a given selection.

The degree of enrichment at which correlation breaks down appears tovary with Cre-line. A downside of PCR pool is that there is no way totell whether it or synthetic pool is the more ‘true’ enrichment score oreven that there may be cause for concern regarding certain enrichmentvalues. The correlation among positively enriched variants between thetwo methods was found to improve with the magnitude of positiveenrichment. For each experiment there is a level of enrichment belowwhich the scores become irreproducible, or noisy. FIG. 50 demonstratesthat neither PCR pool nor Synthetic pool is inherently more ‘true’ atlower enrichment scores. This is because Synthetic pool methodology withits codon replicates has a self-contained control to determine anenrichment level below which enrichment value has no further predictivepower. The term ‘noise’ has been used herein to refer to regions ofenrichment in a particular experiment below which values lose theirreproducibility and predictive power. Being able to experimentallydetermine enrichment signal above noise allows researchers to focustheir attention and data analyses on enrichment levels that areinternally reproducible and thereby avoid selecting false positivevariants or drawing invalid conclusions.

Thus, if one is interested in only the highest enriched variants for aparticular tissue, PCR pool design coupled with enrichment normalizationto virus library may not drastically differ from synthetic pool designover one additional round of selection for a subset of in vivoselections (such as Tek-Cre or SNAP-Cre). Without additional validation,however, it is difficult to predict whether a given in vivo system willperform akin to Tek-Cre. This becomes critical in a multiplexedselection study where target-specific variants may not garner thehighest enrichments in one particular in vivo selection.

Example 5 Analysis of AAV Capsid Libraries After Round-2 Selections

Whereas the AA distribution of the DNA library closely matched theOligopool design, virus production selected for a motif with Asn (N) atposition 2, β-branched AAs (I, T, V) at position 4, and positivelycharged AAs (K, R) at position 5 (FIGS. 10, 52). Fitness for BBBcrossing resulted in a very different pattern. In comparison to the R2virus library, highly enriched variants share preferences, for example,proline (P) in position 5, and phenylalanine (F) in position 6.

The distribution of the positively enriched variants from brain acrossall peripheral organs was then determined (FIG. 11, left). About 60variants that are highly enriched in brain are comparatively depletedacross all other organs (FIG. 11, middle). Encouraged by the expectedbehavior of spike-in control variants (AAV9, PHP.B, PHP.eB), elevennovel variants were chosen for further validation (FIG. 11, right),including several that would have been overlooked if the choice had beenbased on PCR pool or CREATE (Table 8).

TABLE 8 Ranking of AAV-PHP capsids across methods.Ranks of selectedvariants among all capsids recovered from R2 Tek-Cre selection bysynthetic pool enrichment score (representing M-CREATE), PCR poolenrichment score (representing closer to M-CREATE), or PCR pool readcounts (representing CREATE), the highest ranks of which starts from 1,and “Not recovered” represent absence of the variant from R2 sequencingdata. Synthetic pool PCR pool PCR pool AAV enrichment enrichment readcount Variants rank rank rank PHP.V1  1  4  3 PHP.V2  2  1  1 PHP.B4  410  56 PHP.B7  6 13  36 PHP.B8  3  7  23 PHP.C1 13 34  74 PHP.C2 12 20293 PHP.C3 16 Not recovered Not recovered

These variants were chosen due to their enrichments and where they fallin sequence space. The positively enriched variants were found tocluster into distinct families based on sequence similarity. Inagreement with the heatmaps discussed above, the most enriched variantsform a distinct family across selections that share a common motif: T inposition 1, L in position 2, P in positive 5, F in position 6, and K orL in position 7 (FIGS. 12, 53). This AA pattern closely resembles thepreviously identified variant, AAV-PHP.B—TLAVPFK. Given the sequencesimilarity among members, we predicted that they may similarly cross theBBB and target the central nervous system.

The ability to twice recover the AAV-PHP.B sequence family fromcompletely independently constructed and selected libraries confirmsthat the viral library's sequence space coverage was broad enough torecover a family of variants sharing a common motif. Unlike CREATE whichidentified only one variant, AAV-PHP.B, M-CREATE yielded a diversePHP.B-like family that hints toward important chemical features of thismotif. The sequence diversity within this family suggests that isolatingAAV-PHP.B was not simply good fortune in previous experiments(considering a theoretical starting library size of ˜1.3 billion), andthat this is a dominant family for this particular experiment.

Example 6 Capsid Recovery from Round-2 Selection Yields a Pool of AAV9Variants with Enhanced BBB Entry and CNS Ttransduction

Given the dominance of the PHP.B-family in this particular selection,the most enriched member was tested: TALKPFL (FIGS. 12 and 13)henceforth referred to as AAV-PHP.V1. Somewhat surprisingly given itssequence similarity to AAV.PHP.B, the tropism of AAV-PHP.V1 is biasedtoward transducing brain vascular cells (FIGS. 14, 54). When deliveredintravenously, AAV-PHP.V1 carrying a fluorescent reporter under thecontrol of the ubiquitous CAG promoter transduces ˜60% of GLUT1⁺cortical brain vasculature compared to ˜20% with AAV-PHP.eB and almostno transduction with AAV9 (FIGS. 14 and 16). In addition to thevasculature, AAV-PHP.V1 also transduced ˜60% of cortical S100⁺astrocytes (FIG. 17). However, AAV-PHP.V1 is not as efficient forastrocyte transduction as the previously reported AAV-PHP.eB (whenpackaged with an astrocyte specific GfABC1D promoter, FIG. 55).

For applications requiring endothelial cell-restricted transduction viaintravenous delivery, AAV-PHP.V1 vectors can be used in three differentsystems: (1) in endothelial cell-type specific Tek-Cre mice with aCre-dependent expression vector (FIG. 15 (left) and FIG. 18), (2) influorescent reporter mice where Cre is delivered with an endothelialcell-type specific MiniPromoter (Ple261) (FIG. 15 (right) and FIGS. 19and 56 through the left column of FIGS. 58), and (3) in wild-type miceby packaging a self-complementary genome (scAAV) containing a ubiquitouspromoter (Right column of FIG. 58). The mechanism of endothelialcell-specific transduction by AAV-PHP.V1 using scAAV genomes is unclear,but shifts in vector tropism when packaging scAAV genomes have beenreported for another capsid.

Given the dramatic difference in tropism between AAV-PHP.V1 andAAV-PHP.B/eB, we tested several additional variants within thePHP.B-like family. One variant, AAV-PHP.V2—TTLKPFL, differed by only oneAA from AAV-PHP.V1, has a similar tropism (FIGS. 59-61). AAV-PHP.V2 wasfound at high abundance in R1 selection across all brain libraries andwas highly enriched in R2 (FIGS. 4, 11 (right panel), 12, 13, and 40).Given its sequence similarity, similar tropism was expected to that ofAAV-PHP.V1. This was validated in vivo in C57BL/6J adult mice(ssAAV-PHP.V2:CAG-mNeongreen genome, 3×10¹¹ vg dose per adult mice, n=3,FIG. 59), in Tek-Cre mice (ssAAV-PHP.V2:CAG-DIO-EYFP genome, 1×10¹² vgdose per adult mouse, n=2, FIG. 60), and in GFAP-Cre mice(ssAAV-PHP.V2:CAG-DIO-EYFP, 1×10¹² vg dose per adult mouse, n=2, FIG.61).

Three other variants with sequences of roughly equal deviation from bothAAV.PHP.V1 and AAV.PHP.B, AAV-PHP.B4—TLQIPFK, AAV-PHP.B7—SIERPFK, andAAV-PHP.B8—TMQKPFI (FIGS. 12, 13, 20, and 21), have PHP.B-like tropismwith biased transduction toward neurons and astrocytes (FIGS. 21 and62-64). Similar variants among the spike-in library, AAV-PHP.B5—TLQLPFKand AAV-PHP.B6—TLQQPFK, also shared this tropism (FIGS. 13, 20, 21, and62).

To evaluate the performance of the spike-in library, two highly enrichedvariants similarly placed in sequence space were chosen:AAV-PHP.B6—TLQLPFK and AAV-PHP.B7—TLQQPFK (FIGS. 48 (middle panel), and53) that were previously identified in the 3-mer-s PHP.B library butnever validated in vivo. At a modest dose of 1×10¹¹ vg in C57BL/6J adultmice, these variants also display PHP.B-like tropism (FIGS. 20-21 and62).

A series of variants selected to verify M-CREATE's predictive poweroutside this family were then investigated: (1) A highly enrichedvariant with a completely unrelated sequence, AAV-PHP.C1—RYQGDSV (FIGS.12, 13, 20, and 21), transduced astrocytes at a similar efficiency andneurons at lower efficiency compared to other tested variants fromB-family (FIG. 21). (2) Two variants found in high abundance in the R2synthetic pool virus library and negatively enriched in brain (with bothcodon replicates in agreement), AAV-PHP.X1—ARQMDLS andAAV-PHP.X2—TNKVGNI (FIG. 46, right), poorly transduced the CNS (FIG.63). (3) Two variants that were found in higher abundance in brainlibraries from the PCR pool R2, AAV-PHP.X3—QNVTKGV andAAV-PHP.X4—LNAIKNI also failed to outperform AAV9 in the brain (FIG.65).

Collectively, the characterization of these AAV variants demonstratesseveral key points. First, within a diverse sequence family, there isroom for both functional redundancy and the emergence of novel tropisms.Second, highly enriched sequences outside the dominant family are alsolikely to possess enhanced function. Third, buoyed by codon replicateagreement in the synthetic pool, a variant's enrichment across tissuesmay be predictive. Fourth, while the synthetic pool R2 library containsa subset of the sequences that are in the PCR pool R2 and may therebylack some enhanced variants, the excluded PCR pool population isenriched in false positives.

The ability to confidently predict in vivo transduction from a pool of18,000 variants across mice is a significant advance in the selectionprocess and demonstrates the power of M-CREATE for the evolution ofindividual vectors.

Example 7 Re-Investigation of Capsid Selection that Yielded AAV.PHP.eBReveals Variant that Specifically Transduces Neurons

Using NGS, a 3-mer-s (s-substitution) PHP.B library generated by theprior CREATE methodology that yielded AAV-PHP.eB²⁷ was reinvestigated(FIG. 24). The brain libraries were deep sequenced using Cre-dependentPCR and a R2 liver library from wild-type mice (processed via PCR forall capsid sequences regardless of Cre-mediated inversion) andidentified 150-200 positively enriched capsids in brain tissue (FIGS.25, 66, and 67). Briefly, the re-investigated 3-mer-s PHP.B librarydiversified positions 587-597 of the AAV-PHP.B capsid (equivalent of587-590 AA on AAV9) in portions of three consecutive AAs, (40,000 totalvariants) (FIG. 24). Selections were performed in three Cre-transgeniclines: Vglut2-IRES-Cre for glutamatergic neurons, Vgat-IRES-Cre forGABAergic neurons, and GFAP-Cre for astrocytes.

Variants that were positively enriched in brain and negatively enrichedin liver show a significant bias towards certain AAs: G, D, E atposition 1; G, S at position 2 (which includes the AAV-PHP.eB motif,DG); and S, N, P at position 9, 10, 11 (FIGS. 26 and 68). Variants thatwere positively enriched in the brain were clustered according to theirsequence similarities and ranked by their negative enrichment in liver(represented by node size in clusters). A distinct family referred to asN emerged with a common motif “SNP” at positions 9-11 on PHP.B backbone(FIGS. 27 and 69).

The core variant of the N-family cluster: AQTLAVPFSNP was found in highabundance in R1 and R2 selections, had higher enrichment score in Vglut2and Vgat brain tissues compared to GFAP, and had negative enrichment inliver tissue (FIGS. 25 and 66-69). Unlike AAV-PHP.eB, this variant(AAV-PHP.N) specifically transduced NeuN⁺ neurons even when packagedwith a ubiquitous CAG promoter, although the transduction efficiencyvaried across brain regions (from ˜10-70% in NeuN⁺ neurons, includingboth VGLUT1⁺ excitatory and GAD1⁺inhibitory neurons, FIGS. 28, 29, 70,and 71).

Thus, by re-examining the 3-mer-s library several novel variants wereidentified, including one with notable cell-type-specific tropism. WhileVglut2-Cre and Vgat-Cre mice were used for in vivo selection, novariants stood out for neuronal subtype-specific transduction ofexcitatory and inhibitory populations from initial investigations on theNGS dataset. It is possible that a biological solution to this(stringent) selection was not present in the library.

Example 8 Investigation of Capsid Families Beyond C57BL/6J Mouse Strain

The enhanced CNS tropism of AAV-PHP.eB is absent in a subset of mousestrains. It is highly efficient in C57BL/6J, FVB/NCrl, DBA/2, and SJL/J,with intermediate enhancement in 129S1/SvimJ, and no enhancement inBALB/cJ and several additional strains. This pattern holds for the twonewly identified variants from the PHP.B family, AAV-PHP.V1 andAAV-PHP.N (FIG. 30, Table 9), which did not transduce the CNS inBALB/cJ, yet transduced the FVB/NJ strain (FIG. 31). AAV-PHP.V1transduced Human Brain Microvascular Endothelial Cell (HBMEC) culture,resulting in increased mean fluorescent intensity compared to AAV9 andAAV-PHP.eB (FIG. 72) however, suggesting the potential for mechanisticcomplexity.

TABLE 9 AAV-PHP vectors identified by CREATE and M-CREATE. The tableprovides a summary of the variants that have been identified so farusing CREATE and M-CREATE, along with their tropism and the evolutionarysteps from the parent capsid that was involved in their discovery.Rounds of AAV Reference/ evolution from Variants Selection methodTropism Production parent capsid PHP.B, Deverman et al, Broad CNS Good 1round from B2, B3 2016/CREATE transduction AAV9 PHP.A Deverman et al,Astrocyte Poor; prone to 1 round from 2016/CREATE transductionprecipitate upon AAV9 storage at 4° C. PHP.eB Chan et al, Enhanced BroadGood 2 rounds from 2017/CREATE CNS transduction AAV9 or 1 round fromPHP.B PHP.S Chan et al, Sensory neuron Good 1 round from 2017/CREATEtransduction AAV9 PHP.V1, Current study/ BBB Vascular Good 1 round fromV2 M-CREATE cells and AAV9 astrocytes transduction PHP.B4, Currentstudy/ Broad CNS Good 1 round from B7, B8, M-CREATE transduction AAV9PHP.B5, Current study/ Broad CNS Good 2 rounds from B6 M-CREATE andtransduction AAV9 or CREATE 1 round from PHP.B PHP.C1, Current study/Broad CNS Good; PHP.C1 1 round from C2, C3 M-CREATE transduction proneto AAV9 across mouse precipitate upon strains storage at 4° C. PHP.NCurrent study/ Neuron Average 2 rounds from M-CREATE and transductionAAV9 or CREATE 1 round from PHP.B

Importantly, M-CREATE revealed many non-PHP.B-like sequence familiesthat enriched through selection for transduction of cells in the CNS. Wetested the previously mentioned AAV-PHP.C1: RYQGDSV, as well asAAV-PHP.C2: WSTNAGY, and AAV-PHP.C3: ERVGFAQ (FIG. 30). These showedenhanced BBB crossing irrespective of mouse strain, with roughly equalCNS transduction in BALB/cJ and C57BL/6J (FIGS. 32 and 73).Collectively, these preliminary studies suggest that M-CREATE is capableof finding capsid variants with diverse mechanisms of BBB entry thatlack strain-specificity.

Example 8 Exemplary Insertion of Variant AAV Capsid Protein Sequence

Demonstration of 7 amino acid peptide insertion in AAV capsid: Peptidesequence “TLQIPFK” (SEQ ID: 435) is positioned between AA 588-589 of AAVcapsid. The insertion sequence is underlined and bold.

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQ

AQAQT GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

Demonstration of 11 amino acid peptide insertion in AAV capsid:

Peptide sequence “DGTTLKPFLAQ” (SEQ ID: 867) is positioned by replacingAA 587-590 of AAV capsid. The inserted sequence is underlined andhighlighted.

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQS

AQT GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL.

Example 9 Treating Huntington's Disease (HD)

A subject having Huntington's disease is identified. The subject is thensystemically administered a first amount of a viral vector that includesa polynucleotide that encodes for a Zinc finger protein (ZFP) engineeredto represses the transcription of the Huntingtin (HTT) gene. The vectorwill be encapsidated by a modified AAV capsid protein with an amino acidsequence provided in FIG. 33 or provided in Tables 2-4, so as to allowproper targeting of the ZFP to the nervous system, among other organs.If needed, the subject is administered a second or third dose of thevector, until a therapeutically effective amount of the ZFP is expressedin the subject in the nervous system.

Example 10 Phase 1A Clinical Trial

A phase 1A clinical trial is performed to evaluate the safety,tolerability, pharmacokinetics, and pharmacodynamics of an one-timeintravenous injection of test composition comprising viral vectorencapsidated by a modified capsid protein with any of the amino acidsequences provided in FIG. 33 or Tables 2-4, in subjects with lateHuntington's Disease (HD). Eligible subjects are men and women between21 and 65 years of age.

Inclusion Criteria: Eligible subjects are men and women between 21 and65 years of age. Subjects that (i) sign and date Sign and dateInternational Classification of Functioning, Disability and Health(ICF); (2) male or female participant aged ≥21 and ≤65; (3) participantswho submit medical report (PCR) attesting Huntington's disease with anumber of CAG repeats on chromosome 4, greater than or equal to 40 andless than or equal to 50 (if the participant has not performed theexamination and/or if he does not have the report available, a new examshould be done); (4) Score 5 points or more in motor assessment UHDRSscale (Unified Huntington's Disease Rating Scale) at the time ofenrollment; (5) Score between 8 and 11 points in the functional capacityof the UHDRS scale at the time of enrollment.

Exclusion Criteria: (1) Any medical observation data (clinical andphysical) that medical research judge as a risk for subject ifenrollment at the study; (2) any laboratory exam data that medicalresearch judge as a risk for subject if enrollment at the study; (4)history of epilepsy; (5) diagnostic of major cognitive impairment; (6)active decompensated psychiatric disease; (7) current or prior historyof neoplasia; (8) current history of gastrointestinal, hepatic, renal,endocrine, pulmonary, hematologic, immune, metabolic pathology or severeand uncontrolled cardiovascular disease; (8) diagnostic of any activeinfection, be it viral, bacterial, fungal, or caused by anotherpathogen; (9) participants who have contraindication to undergo any ofthe tests performed in this study, for example, have pacemakers orsurgical clip; (10) history of alcohol or illegal drugs abusers; (11)history of 1 or more episodes of suicide in the two years before VisitV-4; (12) active smoker or have stopped smoking less than six monthsprior to enrollment; (13) test positive in at least one of theserological tests: HIV 1 and 2 (Anti-HIV-1,2), HTLV I and II, HBV(HBsAg, anti-HBc), HCV (anti-HCV-Ab) and VDRL (Treponema pallidum); (14)history of drug allergy, including contrasts for imaging, or bovineproducts; (15) in use or expected use of immunosuppressive drugs orprohibited medicines for the first three months after the firstadministration of the investigational product; (16) any clinical changesthat is interpreted by the medical researcher as a risk to participant'senrollment.

Experimental:

Placebo. One-time injection of placebo at Week 0.

Test High Dose. One-time injection of test composition 2×10{circumflexover ( )}10 vg at Week 0.

Test Middle Dose. One-time injection of test composition 6×10{circumflexover ( )}9 vg at Week 0.

Test Low Dose. One-time injection of test composition 2×10{circumflexover ( )}9 vg at Week 0.

Test Lowest Dose. One-time injection of test composition 2×10{circumflexover ( )}8 vg at Week 0.

Primary Outcome Measures: Safety of the test composition by periodicmonitoring changes at adverse events, vital signs, laboratory tests, ECGand incidence of benign and malignant neoplasms [ Time Frame: five years]. The safety of the investigational product will be evaluated in detailfrom periodic evaluations contemplating monitoring changes of: (1)adverse events including type, frequency, intensity, seriousness,severity, and action taken related to the investigational product study;(2) vital signs (BP, HR, axillary temperature), physical and medicalexamination (BMI, weight, height, medical condition—cardiovascular,pulmonary, digestive, musculoskeletal and peripheral, with emphasis onthe neurological assessment and others); (2) laboratory tests includedhematologic, biochemical, urologic and serological analysis; (3)electrocardiogram (ECG) of 12 derivations; (4) and incidence andclassification of benign and malignant neoplasms in the followingorgans/systems: CNS, lung, liver, spleen, pancreas, prostate, testicle,urinary, hematological and skeletal system through the laboratory tests,magnetic resonance imaging, computerized tomography and ultrasonography.

Secondary Outcome Measures: Preliminary efficacy of Cellavita HD byglobal clinical response (CIBIS) and UHDRS improvement [ Time Frame:five years ] will be evaluated by statistical comparison of the resultsof each UHDRS scale component: motor, cognitive and behavior. The globalclinical response will be assessed by statistical comparison betweenbaseline score observed by the Investigator before and after CellavitaHD treatment. Preliminary efficacy of Cellavita HD by comparison of theinflammatory markers [ Time Frame: one year ] will be evaluated bystatistical comparison of the inflammatory markers included IL-4, IL-6,IL-10 (interleukin IL) and TNF-alpha (tumoral necrosis factor alpha).Immunological Response of Cellavita HD [Time Frame: one year]. Theimmunological response induced by Cellavita HD will be evaluated bystatistical comparison between baseline results of CD4+ and CD8+proliferation and the other evaluated times. Preliminary efficacy ofCellavita HD by comparison of the CNS assessment [Time Frame: one year].Will be evaluated by statistical comparison of the CNS assessmentthrough magnetic resonance image at cortical thickness measurements,volumes of different brain structures, especially the basal ganglia,with special attention to caudate and metabolic changes identified inproton spectroscopy. Risk of suicidal ideation by Hamilton DepressionRating Scale (HDRS) [Time Frame: five years] will be evaluated bysuicidal domain. The classificatory pontuation may correspond to milddepression (score: 8 to 13), moderate depression (score: 19-22) andsevere depression (score: >23).

While preferred instances of the present examples have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch instances are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the disclosure. It should be understood thatvarious alternatives to the instances of the disclosure described hereinmay be employed in practicing the disclosure. It is intended that thefollowing claims define the scope of the disclosure and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. An AAV capsid comprising: a) an AAV capsid protein comprising: i. afirst amino acid sequence that is at least 98% identical to amino acid217 to amino acid 736 of SEQ ID NO: 1; and ii. a second amino acidsequence at least 57.1% identical to an amino acid sequence provided inTables 2-3 or FIG. 33 inserted at an amino acid position 588_589 withinSEQ ID NO: 1, wherein the AAV capsid protein is characterized by atleast one of an increased specificity and an increased transductionefficiency when measured in a central nervous system (CNS) in a subjectwhen delivered to the subject systemically, relative to a native AAVcapsid protein provided in SEQ ID NO:
 1. 2. The AAV capsid of claim 1,wherein the second amino acid sequence is at least 71.4% identical tothe amino acid sequence provided in Tables 2-3 or FIG.
 33. 3. The AAVcapsid of claim 1, wherein the second amino acid sequence is at least86.7% identical to the amino acid sequence provided in Tables 2-3 orFIG.
 33. 4. The AAV capsid of claim 1, wherein the second amino acidsequence is selected from the group consisting of TALKPFL, TTLKPFL,TLQIPFK, TMQKPFI, SIERPFK, RYQGDSV, and TTLKPFS.
 5. The AAV capsid ofclaim 1, wherein the AAV capsid protein is present in VP1, VP2, and VP3of the AAV capsid.
 6. The AAV capsid of claim 1, wherein the AAV capsidis chimeric.
 7. The AAV capsid of claim 1, wherein 60 copies of the AAVcapsid protein are assembled into the AAV capsid.
 8. The AAV capsidprotein of claim 1, wherein the CNS comprises a cell-type selected fromthe group consisting of a neuron, an oligodendrocyte, an astrocyte, anda brain vascular cell.
 9. The AAV capsid of claim 1, wherein the CNScomprises a tissue that is selected from the group consisting of abrain, a thalamus, a cortex, a striatum, a ventral midbrain, and aspinal cord.
 10. The AAV capsid of claim 1, wherein the AAV capsidprotein further comprises an amino acid substitution A587D or Q588G. 11.The AAV capsid protein of claim 1, wherein the AAV capsid proteinfurther comprises an amino acid substitution A589N or Q590P.
 12. The AAVcapsid of claim 1, wherein the second amino acid sequence at the aminoacid position 588_589 within SEQ ID NO: 1 is not TLAVPFK, KFPVALT,SVSKPFL, FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, or YTLSQGW.
 13. The AAVcapsid of claim 1 that is isolated and purified.
 14. The AAV capsid ofclaim 1 formulated as a pharmaceutical formulation for intravenousadministration to treat a disease or a condition of the CNS, thepharmaceutical formulation further comprising a pharmaceuticallyacceptable carrier.
 15. The AAV capsid of claim 14, wherein thepharmaceutical formulation further comprises a therapeutic agent.16.-44. (canceled)
 45. A method of treating a disease or condition in asubject comprising administering a therapeutically effective amount of apharmaceutical formulation comprising an AAV capsid protein comprising:i. a first amino acid sequence that is at least 98% identical to aminoacid 217 to amino acid 736 of SEQ ID NO: 1; and ii. a second amino acidsequence at least 57.1% identical to an amino acid sequence provided inTables 2-3 or FIG. 33 inserted at an amino acid position 588 589 withinSEQ ID NO: 1, wherein the AAV capsid protein is characterized by atleast one of an increased specificity and an increased transductionefficiency when measured in a central nervous system (CNS) in a subjectwhen delivered to the subject systemically, relative to a native AAVcapsid protein provided in SEQ ID NO:
 1. 46. (canceled)
 47. (canceled)48. A method of manufacturing a recombinant AAV particle, the methodcomprising: a) providing a recombinant AAV genome comprising: i. an AAVcapsid gene, and ii. a recognition sequence for a Cre recombinase,wherein the recognition sequence facilitates a recombinase-dependentchange that is detectable, and wherein the recombinase recognitionsequence comprises two Cre-recognition sites; b) transfecting apopulation of cells expressing the Cre recombinase with the recombinantAAV genome, whereby the Cre recombinase induces a recombination event togenerate the recombinase-dependent change in the recombinant AAV genome,and wherein the recombinase-dependent change comprises an inversion ofthe sequence that is flanked by the Cre-recognition sites; c) detectingan increased rate of the recombinase-dependent change a target cell inthe population of cells; d) detecting a decreased rate of therecombinase-dependent change in an off-target cell in the population ofcells; and e) identifying a recombinant AAV genome generated by therecombinase-dependent change, wherein said identified rAAV genomecomprises the inversion, and wherein said identified recombinant AAVgenome encodes an AAV capsid particle characterized having an increasedspecificity for the target cell and a decreased specificity for theoff-target cell.
 49. The method of claim 48, wherein the off-target cellis a hepatocyte.
 50. The method of claim 48, wherein the target cell isa cell selected from the group consisting of a neuron, a glial cell, anoligodendrocyte, an ependymal cell, an astrocyte, a Schwann cell, asatellite cell, and an enteric glial cell.